Preventive and/or therapeutic drugs for asthma

ABSTRACT

The present invention provides an agent for prevention and/or treatment of asthma comprising a substance capable of suppressing the function involved in signal transduction of GPR4 as an active ingredient. It also provides an agent for prevention and/or treatment of asthma which comprises a nitrogen-containing tricyclic compound represented by the formula (I) or a quaternary ammonium salt thereof, or a pharmaceutically acceptable salt thereof;  
                 
 
[wherein R 1  represents a substituted or unsubstituted lower alkyl, or the like; R 2  represents hydrogen, a substituted or unsubstituted lower alkyl, or the like; R 3  and R 4  are the same or different and each represents hydrogen, lower alkyl, or the like; n represents 0 or 1; X represents —(CH 2 ) 2 —, or the like; and Y represents the formula (II)  
                 
 
(wherein W represents CH or a nitrogen atom; Z 1  and Z 2  are the same or different and each represents hydrogen, a substituted or unsubstituted lower alkyl, or the like; and Z 3  represents hydrogen, a substituted or unsubstituted lower alkyl, or the like)] as an active ingredient.

TECHNICAL FIELD

The present invention relates to an agent for prevention and/ortreatment of asthma, which comprises a substance capable of suppressingthe function involved in signal transduction of GPR4 as an activeingredient. The present invention further relates to an agent forprevention and/or treatment of asthma, which comprises anitrogen-containing tricyclic compound or a quaternary ammonium saltthereof, or a pharmaceutically acceptable salt thereof as an activeingredient.

BACKGROUND ART

Bronchial asthma is an inflammatory disease wherein bronchoconstrictionand exacerbation of airway hyperreactivity are main characteristics. Atpresent, daily control of asthma is considered to be fully conducted bya combination of an inhaled steroid, a bronchodilatory drug such as aβ-stimulant or a xanthine-type drug and an anti-allergic drugrepresented by anti-leukotriene drugs. However, steroids which aremainly used for the treatment have side effects and there are patientswho are resistant to steroids or are hardly cured by them, andtherefore, there has been a demand for therapeutic agents having a newmechanism and less side effects.

With regard to GPR4 which is a G-protein coupled-receptor protein(hereinafter, abbreviated as GPCR), it has been known to be highlyexpressed in a lung [Genomics, volume 30, pages 84-88 (1995)]. Inaddition, it has been reported that GPR4 binds to a lipid such assphingosyl phosphorylcholine (SPC) or lysophosphatidyl-choline (LPC) toinduce signals [J. Biol. Chem., volume 276, pages 41325-41335 (2001)].With regard to SPC, it has been reported to induce TNF-α production andICAM-1 expression [J. Invest. Dermatol., volume 112, pages 91-96 (1999)]and it has been suggested that it participates in allergic diseases suchas skin diseases. With regard to LPC, it has been reported that itparticipates in migration of monocytes [Cir. Res., volume 84, pages52-59 (2000)], expression of adhesive molecules in endothelial cells [J.Clin. Invest., volume 90, pages 1138-1144 (1992)], macrophage activation[J. Immunol., volume 147, pages 273-280 (1991).], etc. and participatesin inflammation. Further, with regard to LPC, there are other reportsthat it increases in plasma of patients suffering from asthma [Clinic.Science, volume 97, pages 595-601 (1999)] and that it increases inbronchoalveolar lavage fluid of allergic patients after antigenchallenge [J. Exp. Med., volume 183, pages 2235-2245 (1996)]. There isalso a report that administration of choline which suppresses theproduction of LPC showed a therapeutic effect in patients suffering fromasthma [Indian J. Chest Dis. Allied Sci., volume 39, pages 149-156(1997)]. However, both SPC and LPC have been known to bind to OGR-1[Nat. Cell Biol., volume 2, pages 261-267 (2000)], G2A [Science, volume293, pages 702-705 (2001)], etc. besides GPR4 and it has not been knownwhether such actions are mediated by GPR4.

Among the GPCRs, there has been known a GPCR called a constitutivelyactivated GPCR, which transduces signals even in the absence of a ligandwhen it is excessively expressed in cells. A signal which is transducedin the absence of a ligand is called a constitutive activity. Among theconstitutively activated GPCRs, there are those which are present innature and those which are mutated by introducing a mutation such assubstitution, deletion, etc. of amino acids [Molecular Pharmacology,volume 57, page 890 (2000); WO 98/46995]. An antagonist which suppressesthe constitutive activity of GPCR is called an inverse agonist.

In the literatures [Bulletin de la Société Chimique, page 185 (1981) andEuropean Journal of Medicinal Chemistry, volume 12, page 219 (1977)],compounds wherein R¹ represents morpholino,

-   R², R³ and R⁴⁴ represent hydrogen, a substituent corresponding to Y    is morpholino, n is 1, and X represents —(CH₂)₂— in the formula (I)    which is described later are disclosed.

DISCLOSURE OF THE INVENTION

An object of the present invention is:

1) to provide an agent for prevention and/or treatment of asthma, whichcomprises, as an active ingredient, a substance capable of suppressingthe function involved in signal transduction of GPR4, and

2) to provide an agent for prevention and/or treatment of asthma, whichcomprises, as an active ingredient, a nitrogen-containing tricycliccompound or a quaternary ammonium salt thereof, or a pharmaceuticallyacceptable salt thereof.

The present invention relates to the following (1) to (8).

(1) An agent for prevention and/or treatment of asthma, which comprises,as an active ingredient, a substance capable of suppressing the functioninvolved in signal transduction of a protein having the amino acidsequence represented by SEQ ID NO: 11.

(2) An agent for prevention and/or treatment of asthma, which comprisesone of the following 1) to 4) as an active ingredient:

1) an oligonucleotide having a sequence complementary to that ofoligonucleotide comprising continuous 5 to 60 nucleotides selected fromthe nucleotide sequence represented by SEQ ID NO: 12 or a derivative ofsaid oligonucleotide,

2) an oligonucleotide having a sequence complementary to that ofoligonucleotide comprising continuous 5 to 60 nucleotides selected fromthe nucleotide sequence represented by SEQ ID NO: 14 or a derivative ofsaid oligonucleotide,

3) an oligonucleotide having a sequence complementary to that ofoligonucleotide comprising continuous 5 to 60 nucleotides selected fromthe nucleotide sequence represented by SEQ. ID NO: 18 or a derivative ofsaid oligonucleotide, and

4) an oligonucleotide comprising 5 to 60 nucleotides which hybridizesunder stringent conditions with DNA having the nucleotide sequencerepresented by one member selected from SEQ ID NOs: 12, 14 and 18 andwhich is capable of suppressing the function involved in signaltransduction of protein having the amino acid sequence represented bySEQ ID NO: 11 or a derivative of said oligonucleotide.

(3) An agent for prevention and/or treatment of asthma, which comprisesone of the following 1) to 4) as an active ingredient:

1) an antibody which recognizes a protein having the amino acid sequencerepresented by SEQ ID NO: 11,

2) an antibody which recognizes a protein having the amino acid sequencerepresented-by-SEQ ID NO: 13,

3) an antibody which recognizes a protein having the amino acid sequencerepresented by SEQ ID NO: 17, and

4) an antibody, which recognizes a protein having the amino acidsequence in which one or more amino acid(s) is/are deleted, substitutedor added in the amino acid sequence represented by one member selectedfrom SEQ ID NOs:11, 13 and 17 and which has the function involved insignal tranduction of a protein having the amino acid sequencerepresented by SEQ ID NO:11

(4) An agent for prevention and/or treatment of asthma, which comprisesa nitrogen-containing tricyclic compound represented by the formula (I)or a quaternary ammonium salt thereof, or a pharmaceutically acceptablesalt thereof;

[wherein R¹ represents a substituted or unsubstituted heterocyclicgroup, —NR⁵R⁶ (wherein R⁵ and R⁶ are the same or different and eachrepresents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aralkyl or substituted or unsubstituted heterocyclicalkyl, or R⁵ and R⁶ are combined together with the adjacent-nitrogenatom to form a substituted or unsubstituted heterocyclic group), —OR⁷(wherein R⁷ represents hydrogen, substituted or unsubstituted loweralkyl, substituted or unsubstituted lower alkanoyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted lower alkenyl,substituted or unsubstituted lower alkynyl, substituted or unsubstitutedaryl, substituted or unsubstituted aralkyl or substituted orunsubstituted heterocyclic alkyl), —SR^(7a) (wherein R^(7a) has the samemeaning as the above R⁷), —CONR^(5a)R^(6a) (wherein R^(5a) and R^(6a)have the same meanings as the above R⁵ and R⁶ respectively), —CO₂R^(7b)(wherein R^(7b) has the same meaning as the above R⁷), —N⁺R^(5b)R^(6b)R⁸(wherein R^(5b) and R^(6b) have the same meanings as the above R⁵ andR⁶, respectively, and R⁸ represents lower alkyl, lower alkenyl oraralkyl), formyl, carboxy or cyano;

R² represents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aralkyl or substituted or unsubstituted heterocyclicalkyl;

R³ and R⁴ are the same or different and each represents hydrogen, loweralkyl or halogen;

n represents 0 or 1;

X represents —(CH₂)₂— or —CH═CH—; and

Y represents the formula (II);

(wherein W represents CH or a nitrogen atom;

Z¹ and Z² are the same or different and each represents hydrogen,substituted or unsubstituted lower alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted lower alkenyl, substituted orunsubstituted lower alkynyl, substituted or unsubstituted aryl,substituted or unsubstituted aralkyl or substituted or unsubstitutedheterocyclic alkyl, or Z¹ and Z² are combined together with two carbonatoms being adjacent to each of them to form a substituted orunsubstituted aromatic ring or substituted or unsubstituted heterocycle;and

Z³ represents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aryl, a substituted or unsubstituted heterocyclicgroup, substituted or unsubstituted aralkyl or substituted orunsubstituted heterocyclic alkyl)] as an active ingredient.

(5) The agent for prevention and/or treatment of asthma according to(4), wherein R¹ is —NR⁵R⁶ and R⁵ and R⁶ are combined together with theadjacent nitrogen atom to form a substituted or unsubstitutedheterocyclic group.

(6) The agent for prevention and/or treatment of asthma according to (4)or (5), wherein R² is hydrogen.

(7) The agent for prevention and/or treatment of asthma according to anyone of (4) to (6), wherein R³ and R⁴ are hydrogen.

(8) The agent for prevention and/or treatment of asthma according to anyone of (4) to (7), wherein Z¹ and Z² are combined together with twocarbon atoms being adjacent to each of them to form substituted orunsubstituted heterocycle;

(9) A method for prevention and/or treatment of asthma, which comprisesadministering an effective amount of the nitrogen-containing tricycliccompound or the quaternary ammonium salt thereof, or thepharmaceutically acceptable salt thereof described in any one of (4) to(8).

(10) Use of the nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofdescribed in any one of (4) to (8) for the manufacture of an agent forprevention and/or treatment of asthma.

The present invention further relates to the following (11) to (23).

(11) A nitrogen-containing tricyclic compound represented by the formula(I) or a quaternary ammonium salt thereof, or a pharmaceuticallyacceptable salt thereof;

(wherein n, R¹, R², R³, R⁴, X and Y each have the same meanings asdefined above, respectively).

(12) The nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofaccording to (11), wherein R¹ is —NR⁵R⁶ and R⁵ and R⁶ are combinedtogether with the adjacent nitrogen atom to form a substituted orunsubstituted heterocyclic group.

(13) The nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofaccording to (11) or (12), wherein R² is hydrogen.

(14) The nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofaccording to any one of: (11) to (13), wherein R³ and R⁴ are hydrogen.

(15) The nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofaccording to any one of (11) to (14), wherein Z¹ and Z² are combinedtogether with two carbon atoms being adjacent to each of them to formsubstituted or unsubstituted heterocycle.

(16.) A pharmaceutical composition comprising the nitrogen-containingtricyclic compound or the quaternary ammonium salt thereof, or thepharmaceutically acceptable salt thereof according to any one of (11) to(15) as an active ingredient.

(17) A suppressor of the function involved in signal transduction of aprotein having the amino acid sequence represented by SEQ ID NO: 11,comprising the nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofaccording to any one of (11) to (15) as an active ingredient.

(18) A method for prevention and/or treatment of asthma, which comprisesadministering a therapeutically effective amount of a substance capableof suppressing the function involved in signal transduction of a proteincomprising the amino acid sequence represented by SEQ ID NO: 11.

(19) A method for prevention and/or treatment of asthma, which comprisesadministering a therapeutically effective amount of an oligonucleotideor a derivative of said oligonucleotide which is any one of 1) to 4)described in (2).

(20) A method for prevention and/or treatment of asthma, which comprisesadministering a therapeutically effective amount of an antibody which isany one of 1) to 4) described in (3).

(21) Use of a substance capable of suppressing the function involved insignal transduction of a protein having the amino acid sequencerepresented by SEQ ID NO: 11 for the manufacture of an agent forprevention and/or treatment of asthma.

(22) Use of an oligonucleotide or a derivative of said oligonucleotidewhich is any one of 1) to 4) described in (2) for the manufacture of anagent for prevention and/or treatment of asthma.

(23) Use of an antibody which is any one of 1) to 4) described in (3)for the manufacture of an agent for prevention and/or treatment ofasthma.

Thus, according to the present invention, there is provided a novelnitrogen-containing tricyclic compound or a quaternary ammonium saltthereof, or a pharmaceutically acceptable salt thereof described in (11)to (15) and there are provided a pharmaceutical composition comprisingthe same as an active ingredient and a suppressor of the functioninvolved in signal transduction of a protein comprising the amino acidsequence represented by SEQ ID NO:11.

The present inventors have found a new, finding that a substance capableof suppressing the function involved in signal transduction of GPR4which belongs to GPCRs is effective for prevention and/or treatment ofasthma and have achieved the present invention. The present inventorshave searched substances capable of suppressing the constitutiveactivity of GPR4 which belongs to constitutively activated GPCRs andhave found that a substance capable of suppressing the constitutiveactivity of GPR4 is effective for prevention and/or treatment of asthma.

Substances capable of suppressing the function involved in signaltransduction of GPR4 include a substance capable of inhibiting orsuppressing the expression of GPR4 itself, a substance capable ofinhibiting the binding of a ligand to GPR4, a substance capable ofsuppressing signal transduction caused by the binding of a ligand toGPR4 [such as changes (rise or fall) in intracellular cAMPconcentration, changes (rise) in intracellular Ca²⁺ concentration andphosphorylation of mitogen-activated protein (MAP)-kinase], a substancecapable of suppressing signal transduction-caused by a constitutiveactivity of GPR4 (such as an inverse agonist of GPR4), etc. There is noparticular limitation for the structure for the above substances so faras they carry such functions, and substances having a known structuremay be acceptable as well. Examples of GPR4 are a protein having theamino acid sequence represented by any one selected from SEQ ID NOs: 11,13 and 17, a protein having the amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted or added in the amino acidsequence represented by any one selected from SEQ ID NOs: 11, 13 and 17and which has the function involved in signal transduction of a proteinhaving the amino acid sequence represented by SEQ ID NO: 11, etc.

The protein having an amino acid sequence wherein one or more aminoacid(s) are/is deleted, substituted or added in the amino acid sequencerepresented by any one selected from SEQ ID NOs: 11, 13 and 17 and alsohaving a function involved in the signal transduction of protein havingthe amino acid sequence represented by SEQ ID NO: 11 can be obtained,for example, by introducing a site-specific mutation into DNA encoding aprotein having an amino acid sequence represented by any one selectedfrom SEQ ID NO: 11, 13 and 17 by site-directed mutagenesis described inthe literatures [Molecular Cloning, A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press (1989) (hereinafter, abbreviated as“Molecular Cloning, Second Edition”); Current Protocols in MolecularBiology, John Wiley & Sons (1987-1997) (hereinafter, abbreviated as“Current Protocols in Molecular Biology”); Nucleic Acids Research, 10,6487 (1982); Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315(1985); Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci.USA, 82, 488 (1985); etc.].

Although there is no particular limitation for the number(s) of aminoacid(s) which is/are deleted, substituted or added, it/they is/are from1 to several tens, preferably from 1 to 20, more preferably 1 to 10 and,still more preferably, 1 to 5.

The expression “one or more amino acid(s) are/is deletion; substitutionor addition in the amino acid sequence represented by any one selectedfrom SEQ ID NO: 11, 13 and 17” means that the amino acid sequence maycontain deletion, substitution or addition of single or plural aminoacid residue(s) at an arbitrary position therein. Deletion, substitutionor addition may be simultaneously contained in one sequence and, theamino acid residue(s) to be deleted, substituted or added, may be eithernatural or non-natural. Examples of natural amino acid residues areL-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine and L-cysteine.

Preferred examples of amino acid residue(s) which is/are mutually ableto be substituted are shown as hereunder. Amino acid residues belongingto the same group are able to be mutually substituted.

Group A: Leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, tert-butylglycine,tert-butylalanine and cyclohexylalanine;

Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid and 2-aminosuberic acid;

Group C: Asparagine and glutamine;

Group D: Lysine, arginine, ornithine, 2,4-diaminobutanoic acid and2,3-diaminopropionid acid;

Group E: Proline, 3-hydroxyproline and 4-hydroxyproline;

Group F: Serine, threonine and homoserine; and

Group G: Phenylalanine and tyrosine.

Also, in order that the protein having an amino acid sequence whereinone or more amino acid residue(s) is/are deleted, substituted or addedin the amino acid sequence represented by any one selected from SEQ IDNO: 11, 13 and 17 has a function involved in the signal transduction ofthe protein having an amino acid sequence represented by SEQ ID NO: 11,it is preferred that said amino acid sequence and the amino acidsequence represented by SEQ ID NO: 11 have a homology of at least 75%,usually not less than 80%, preferably not less than 90% or, morepreferably, not less than 95%.

Homology of amino acid sequence and nucleotide sequence can bedetermined by using-the-algorithm BLAST by Karlin and Altschul [Pro.Natl. Acad. Sci. USA, 90, 5873 (1993)]; or by FASTA [Methods Enzymol.,183, 63 (1990)]. On the basis of the algorithm. BLAST, programs calledBLASTN (database for nucleotide sequence vs. nucleotide sequence) andBLASTX (database for nucleotide sequence vs. amino acid sequence) havebeen developed [J. Mol. Biol., 215, 403 (1990)]. When a nucleotidesequence is analyzed by BLASTN based upon-BLAST, for example, parameterscan be set to score=100 and wordlength=12. When an amino acid sequenceis analyzed by BLASTX based upon BLAST, for example, parameters can beset to score=50 and wordlength=3. When BLAST and Gapped BLAST programsare used, a default parameter for each program can be used. [With regardto Gapped BLAST, refer to the literature (Nuc. Acids Res., 25, 3389-3402(1997)).] Specific means for those analytical methods are known (referto http://www.ncbi.nlm.nih.gov).

Examples of a substance capable of inhibiting or suppressing theexpression of GPR4 itself are an oligonucleotide (hereinafter, referredto as “antisense oligonucleotide”) having a complementary sequence ofoligonucleotide comprising continuous 15 to 60 nucleotides selected fromthe nucleotide sequence represented by any one selected from SEQ ID NO:12, 14 and 18; an oligonucleotide which hybridizes under stringentconditions with DNA having the nucleotide sequence represented by anyone selected from SEQ ID NO: 12, 14 and 18 and suppresses the functioninvolved in signal transduction of protein having the amino acidsequence represented by SEQ ID NO: 11; a derivative of theoligonucleotide as such (hereinafter, referred to as “oligonucleotidederivative”); etc.

The antisense oligonucleotide mentioned in the above is not particularlimited so far as it is an antisense oligonucleotide having a sequencecomplementary to an oligonucleotide comprising continuous 15 to 60nucleotides selected from the nucleotide sequences represented by anyone selected from SEQ ID NO: 12, 14 and 18 although preferred one is anantisense oligonucleotide having 17 to 60 nucleotides, more preferably20 to 60 nucleotides and, still more preferably, 30 to 60 nucleotides.Particularly preferred one is an antisense oligonucleotide having acomplementary sequence of translation initiation region of theabove-mentioned oligonucleotide. Said antisense oligonucleotide can beprepared by a conventional method such as by using a DNA synthesizer onthe basis of information concerning a nucleotide sequence for anucleotide sequence represented by any one selected from SEQ ID NOs: 12,14 and 18 or for a nucleotide sequence of fragment thereof.

Examples of the oligonucleotide derivative are an oligonucleotidederivative wherein a phosphoric acid diester bond in an oligonucleotideis converted into a phosphorothioate bond; an oligonucleotide derivativewherein a phosphoric acid diester bond in an oligonucleotide isconverted to an N3′-P5′ phosphoamidate bond; an oligonucleotidederivative wherein a bond of phosphoric acid diester with ribose in anoligonucleotide is converted to a peptide-nucleic acid bond; anoligonucleotide derivative wherein uracil in an oligonucleotide issubstituted with C-5 propynyluracil; an oligonucleotide derivativewherein uracil in an oligonucleotide is substituted with C-5thiazolyluracil; an oligonucleotide derivative wherein cytosine in anoligonucleotide is substituted with C-5 propynylcytosine; anoligonucleotide derivative wherein cytosine in an oligonucleotide issubstituted with a phenoxazine-modified cytosine; an oligonucleotidederivative wherein ribose in an oligonucleotide is substituted with2′-O-propylribose; and an oligonucleotide derivative wherein ribose inan oligonucleotide is substituted with 2′-methoxyethoxyribose [SaiboKogaku, 16, 1463 (1997)].

The expression of GPR4 itself can be inhibited or suppressed by usingthe above-mentioned antisense oligonucleotide or oligonucleotidederivative in accordance with an antisense RNA/DNA technology[Bioscience and Industry, 50, 322 (1992); Kagaku, 46, 681 (1991);Biotechnology; 9, 358 (1992); Trends in Biotechnology, 10, 87 (1992);Trends in Biotechnology, 10, 152 (1992); Saibo Kogaku, 16, 1463 (1997)],a triple helix technology [Trends in Biotechnology, 10, 132 (1992)], aribozyme-technology [Current Opinion in Chemical Biology, 3, 274 (1999);FEMS Microbiology Reviews, 23, 257 (1999); Frontiers in Bioscience, 4,D497 (1999); Chemistry & Biology, 6, R³³ (1999), Nucleic Acids Research,26, 5237 (1998); Trends in Biotechnology, 16, 438 (1998)] or a decoy DNAmethod [Nippon Rinsho—Japanese Journal of Clinical Medicine, 56, 563(1998); Circulation Research, 82, 1023 (1998); Experimental Nephrology,5, 429 (1997); Nippon Rinsho—Japanese Journal of Clinical Medicine, 54,2583 (1996)].

The oligonucleotide which hybridizes under stringent conditions to DNAhaving the nucleotide sequence represented by any one selected from SEQID NOs: 12, 14 and 18 is a DNA which is obtained by colonyhybridization, plaque hybridization, Southern blot hybridization, etc.using a part of or whole DNA having the nucleotide sequence representedby any one selected from SEQ ID NOs: 12, 14 and 18 as a probe. To bemore specific, the DNA includes a DNA which can be, identified bycarrying out hybridization at 65° C. in the presence of 0.7 to 1.0 mol/lof sodium chloride using a filter on which a DNA prepared from coloniesor plaque is immobilized, and then washing the filter under thecondition of 65° C. using an SSC solution of 0.1- to 2-foldconcentration (composition of an SSC solution of a 1-fold concentrationcomprises 150 mmol/l of sodium chloride and 15 mmol/l of sodiumcitrate). Hybridization can be carried out according to a methodmentioned, for example, in Molecular Cloning, Second Edition; CurrentProtocols in Molecular Biology; DNA Cloning 1: Core Techniques, APractical Approach, Second Edition, Oxford University (1995). Theoligonucleotide which is hybridizable includes a DNA having at least 75%homology preferably not less than 80% homology, more preferably not lessthan 95% homology to DNA having a complementary sequence of DNA havingthe nucleotide sequence represented by any one selected from SEQ ID Nos:12, 14 and 18 when calculated by, for example, the above-mentioned BLASTor FASTA. DNA, RNA and the like can be used as the oligonucleotide, andDNA can be used preferably.

A formulation prepared according to the following conventional methodfrom the antisense oligonucleotide or the derivative of said antisenseoligonucleotide described in the above, or the oligonucleotide or thederivative of said oligonucleotide, wherein the oligonucleotidehybridizes under stringent conditions to DNA having the nucleotidesequence represented by any of selected from SEQ ID NOs: 12, 14 and 18,either solely or after inserting into a vector for gene therapy such asa retrovirus vector, an adenovirus vector or an adenovirus associatedvirus vector or the like, also can be used as an agent for preventionand/or treatment of asthma.

When the vector for gene therapy is used as said agent for preventionand/or treatment, it is able to be manufactured by compounding saidvector for gene therapy with a carrier used for gene therapy agent[Nature Genet., 8, 42 (1994)].

With regard to the above carrier, any carrier may be used so far as itis a carrier which is commonly used for injection preparations and itsexamples are distilled water, a salt solution such as sodium chloride ora mixture of sodium chloride or inorganic salt, a solution of saccharidesuch as mannitol, lactose, dextran and glucose, a solution of amino acidsuch as glycine and arginine and a mixed solution of organic acidsolution or a salt solution with glucose solution. It is also possiblethat, in accordance with the common method, an excipient such as osmoticpressure adjusting agent, pH adjusting agent, plant oil such as sesameoil or soybean oil, lecithin or surfactant such as nonionic surfactantis added for the carrier whereupon an injection solution is prepared assolution, suspension or dispersion. It is further possible that such aninjection solution is prepared in a form of being dissolved immediatelybefore use after an operation such as pulverization and freeze-drying.

Said agent for prevention and/or treatment may be used as it is when theagent is liquid or, when the agent is solid, it may be used afterdissolving, immediately before use, in the above-mentioned carrier whichis subjected to a sterilization treatment if necessary.

An example of the method for administration is a local administration sothat it is able to be absorbed with the site of the patient to betreated. It is also possible to transport the DNA to the aimed site forthe treatment by means of a non-viral gene transfection.

Examples of the non-viral gene transfection method are a calciumphosphate-coprecipitation method [Virology, 52, 456-467 (1973); Science,209, 1414-1422 (1980)], a microinjection method [Proc. Natl. Acad. Sci.USA, 77, 5399-5403 (1980); Proc. Natl. Acad. Sci. USA, 77, 7380-7384(1980); Cell, 27, 223-231 (1981); Nature, 294, 92-94 (1981)]; a membranefusion-intervening transfection method using liposome [Proc. Natl. Acad.Sci. USA, 84, 7413-7417 (1987); Biochemistry, 28, 9508-9514 (1989); J.Biol. Chem., 264, 12126-12129 (1989); Hum. Gene Ther., 3, 267-275(1992); Science, 249, 1285-1288 (1990); Circulation, 83, 2007-2011(1992)], a direct DNA-incorporation or receptor-mediated DNAtransfection method [Science, 247, 1465-1468 (1990); J. Biol. Chem.,266, 14338-14342 (1991); Proc. Natl. Acad. Sci. USA, 87, 3655-3659(1991); J. Biol. Chem. 264, 16985-16987 (1989); BioTechniques, 11,474-485 (1991); Proc. Natl. Acad. Sci. USA, 87, 3410-3414 (1990); Proc.Natl. Acad. Sci. USA, 88, 4255-4259 (1991); Proc. Natl. Acad. Sci. USA,87, 4033-4037 (1990); Proc. Natl. Acad. Sci. USA, 88, 8850-8854 (1991);Hum. Gene Ther. 3, 147-154 (1991)], etc.

The substance which inhibits a binding of a ligand to GPR4 includes anantibody which recognizes GPR4, a compound which has an antagonisticaction to GPR4, or the like.

With regard to the above-mentioned antibody, any antibody may be used sofar as it is an antibody which recognizes GPR4 although an antibodywhich specifically recognizes GPR4 is preferred. Said antibody may beeither a polyclonal antibody or a monoclonal antibody. With regard tosuch an antibody, an example is a neutraizing antibody which recognizes.GPR4. A chimera antibody of a human type, a humanized antibody, and thelike also can be used as an antibody of the present invention.

The above-mentioned antibody can, for example, be prepared according tothe following-methods.

(1) Preparation of a Polyclonal Antibody

A polyclonal antibody can be prepared in such a manner that purifiedsample of GPR4 or a partial fragment polypeptide thereof or a peptidehaving a part of amino acid sequence of GPR4 is used as an antigen andadministered to an animal.

With regard to the animal to which administration is conducted, it ispossible to, use rabbit, goat, rat, mouse, hamster, etc.

The amount of said antigen to be adiministered is preferred to be 50 to100 μg per animal.

When a peptide is used, it is preferred to use a product wherein apeptide is subjected to a covalent bond to a carrier protein such askeyhole limpet hemocyanin, bovine thyroglobulin, or the like. A peptideused as an antigen may be synthesized by a peptide synthesizer.

Administration of said antigen is conducted for three to ten times everyone or two week(s) after the first administration. After eachadministration, blood is collected from venous plexus of fundus of theeye on the third to the seventh day and it is confirmed by means of anenzyme-linked immunosorbent assay [ELISA Method: published by IgakuShoin (19.76); Antibodies—A Laboratory Manual, Cold Spring Harbor,Laboratory (1988)] or the like that said serum reacts with an antigenused for immunization.

Serum is obtained from non-human mammals wherein the serum thereof showsa sufficient antibody value to the antigen used for immunization andsaid serum is separated and purified whereupon a polyclonal antibody isable to be prepared.

With regard to a method for the separation and the purification, it ispossible to conduct a treatment by centrifugal separation, salting-outusing 40-50% saturated ammonium sulfate, precipitation with caprylicacid [Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory,(1988)], chromatography using DEAE-Sepharose column, anion exchangecolumn, protein A or G column, gel filtration column or the like, etc.either solely or in combination.

(2) Preparation of Monoclonal Antibody

(a) Preparation of Antibody-Producing Cells

To the pure sample of GPR4 used for immunization or the partial fragmentpolypeptide thereof or to the peptide having an amino acid sequence of apart of GPR4 is used a rat where its serum shows a sufficient antibodyvalue as a supplying source Of antibody-producing cells.

After the third to the seventh day from the final administration of anantigen substance to the rat showing the antibody value, its spleen isexcised.

The spleen is finely cut in an MEM medium (manufactured by NissuiPharmaceutical), loosened using a forceps and centrifuged at 1,200 rpmfor 5 minutes and a supernatant liquid is discarded.

Spleen cells of the resulting precipitate fraction is treated withTris-ammonium chloride buffer (pH 7.65) for 1 to 2 minute(s) to removeerythrocytes and washed with an MEM medium for three times and theresulting spleen cells are used as antibody-producing cells.

(b) Preparation of Myeloma Cells

Established cell line prepared from mouse or rat is used as myelomacell. For example, it is possible to use 8-azaguanine resistant mouse(derived from BALB/c) myeloma cell line P3-X63Ag8-U1 (hereinafter,abbreviated as P3-U1) [Curr. Topics Microbiol. Immunol., 81, 1 (1978);Eur. J. Immunol., 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269(1978)], P3-X63-Ag8653 (653) [J. Immunol., 123, 1548 (1979)], P3-X63-Ag8(X63) [Nature, 256, 495 (1975)], etc. Those cell lines are passagedusing an 8-azaguanine medium [8-azaguanine (15 μg/ml) is further addedto a medium (hereinafter, referred to as “normal medium”) whereinglutamine (1.5 mmol/l), 2-mercaptoethanol (5×10⁻⁵ mol/l), gentamicin (10μg/ml) and fetal bovine serum (FCS) (manufactured by CSL; 10%) are addedto an RPMI-1640 medium]. Thus incubation is conducted on a normal mediumbefore 3 to 4 days of cell fusion and not less than 2×10⁷ of said cellsare used for the fusion.

(c) Preparation of Hybridoma

The antibody-producing cells prepared in (a) and the myeloma cellsprepared in (b) are well washed with an MEM medium or a PBS (1.83 g ofdisodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g-of-sodiumchloride and 1 liter of distilled water; pH 7.2) and mixed so as to makethe ratio of (antibody-producing cells):(myeloma cells) 5 to 10:1, themixture is centrifuged at 1,200 rpm for 5 minutes and a supernatantliquid is discarded.

Cell group of the resulting precipitate fraction is well loosened, 0.2to 1 ml of a solution wherein 2 g of polyethylene glycol (PEG-1000), 2ml of MEM and 0.7 ml of dimethyl sulfoxide (DMSO) are mixed is added tosaid cell group per 10⁸ antibody-producing cells with stirring at 37° C.and then 1 to 2 ml of an MEM medium is added for several times every 1to 2 minute(s).

After addition, an MEM medium is added so as to prepare a solution in anamount of 50 ml. Said prepared solution is centrifuged at 900 rpm for 5minutes and a supernatant liquid is discarded. Cells of the resultingprecipitate fraction are gently loosened and gently suspended in 100 mlof an HAT medium [a medium where hypoxanthine (10⁻⁴ mol/l), thymidine,(1.5×10⁻⁵ mol/l) and aminopterin (4×10⁻⁷ mol/l) are added to a normalmedium] by means of suction and spouting using a measuring pipette.

Said suspension is dispensed in a 96-well incubation plate in an amountof 100 μl per well and is incubated in a 5% CO₂ incubator at 37° C. for7 to 14 days.

After the incubation, a part of a supernatant liquid thereof is takenout and a hybridoma which specifically reacts with the partial fragmentpolypeptide of the polypeptide of the present invention is selected byan enzymatic immunoassay mentioned, for example, in Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988).

The following methods may be listed as specific examples of theenzymatic immunoassay.

In immunization, GPR4 used as antigen or a pure sample of a partialfragment polypeptide thereof or a peptide having a partial amino acidsequence of GPR4 is coated on an appropriate plate, subjected to areaction using supernatant liquid of incubated hybridoma or the pureantibody prepared in (d) which is mentioned later as the first antibody,then subjected to a reaction using anti-rat or anti-mouse immunoglobulinlabeled with biotin, enzyme, chemoluminescent substance or radiationcompound as the second antibody and subjected to a reaction inaccordance with a labeled substance and that which specifically reactswith a polypeptide used as an antigen is selected as a hybridoma whichproduces a monoclonal antibody to be used in the present invention.

Cloning by a limiting dilution method using said hybridoma is repeatedtwice [in the first one, an HT medium (a medium wherein aminopterin isremoved from an HAT medium) is used and, in the second one, a normalmedium is used] and that which shows a strong antibody value in a stablemanner is selected as a hybridoma strain producing a monoclonal antibodyto be used in the present invention.

(d) Preparation of Monoclonal Antibody

The hybridoma cells (5 to 20×10⁶ cells/animal) producing monoclonalantibody to be used in the present invention which are prepared in (c)are intraperitoneally injected to mouse or nude mouse of 8 to 10 weeksage treated with pristane [0.5 ml of pristane(2,6,10,14-tetramethylpentadecane) is intraperitoneally injectedfollowed by breeding for two weeks]. Hybridoma becomes ascites tumorwithin 10 to 21 days.

Ascites is collected from said mouse becoming ascites tumor andcentrifuged at 3,000 rpm for 5 minutes to remove solid.

From the resulting supernatant liquid, monoclonal antibody is able to bepurified and prepared by the method similar to that used for polyclonalantibody.

Determination of subclass of the antibody is carried out using a mousemonoclonal antibody typing kit or a rat monoclonal antibody typing kit.Amount of the polypeptide is calculated by a Lowry method or from theabsorbance at 280 nm.

An agent for prevention and/or treatment of asthma comprising theabove-mentioned antibody capable of recognizing GPR4 is able to beprepared as follows.

With regard to a medicament comprising said antibody as an activeingredient, although it is possible that said active ingredient isadministered solely, it is usually preferred to provide as apharmaceutical preparation by mixing of said active ingredient with oneor more pharmaceutically acceptable carrier(s) followed by manufacturingby any method which is well-known in the technical field ofpharmaceutical preparation sciences. Preferably, an aseptic solutionbeing dissolved in an aqueous carrier such as water or aqueous solutionof sodium chloride, glycine, glucose, human albumin, etc. is used. It isalso possible to add a pharmaceutically acceptable additive such asbuffering or isotonizing agent for making the pharmaceutical preparationsolution nearer the physiological condition such as sodium acetate,sodium chloride; sodium lactate, potassium chloride and sodium citrate.It is further possible to preserve by freeze-drying and to dissolve inan appropriate solvent in actual use.

With regard to the administering route, it is desirable to use thatwhich is most effective for the therapy and its examples are oraladministration and parenteral administration such as intravenousinjection. Examples of the dosage form are tablets, injections, or thelike.

With regard to the preparation suitable for oral administration, tabletsmay be exemplified. They are able to be manufactured using additivesincluding excipient such as lactose and mannitol, disintegrating agentsuch as starch, lubricant such as magnesium stearate, binder such ashydroxypropyl cellulose, surfactant such as fatty acid ester,plasticizer such as glycerol, etc.

With regard to the preparation suitable for parenteral administration,injections may be exemplified. They may be prepared, for example, usinga carrier comprising a salt solution, a glucose solution or a mixturethereof. It is also possible to use the components exemplified asadditives in the oral preparation even in the case of parenteralpreparation.

Dose and administering frequency may vary depending upon aimedtherapeutic effect, administering method, period of treatment, age, bodyweight, etc. and, usually, they are from 10 μg/kg to 8 mg/kg per day foradults.

A substance which is capable of suppressing the function involved insignal transduction resulted from a constitutive activity of GPR4 isalso able to be prepared by investigating the substances which are ableto suppress the signal transduction resulted by said constitutiveactivity.

An example of the compounds having an antagonistic action to GPR4 is acompound represented by the formula (I). Hereinafter, a compoundrepresented by the formula (I) is referred to as compound (I). That isalso applied to compounds having other formula numbers.

In definitions of the groups in compound (I), the followingexemplification is listed.

(i) With regard to the lower alkyl and the lower alkyl moiety in thelower alkanoyl, linear or branched alkyl having 1 to 10 carbon(s) may beexemplified and specific examples thereof are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,neopentyl, hexyl, heptyl, octyl, isooctyl, nonyl and decyl.

(ii) With regard to the cycloalkyl, cycloalkyl having 3 to 8 carbons maybe exemplified and specific examples thereof are cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

(iii) With regard to the lower alkenyl, linear, branched or cyclicalkenyl having 2 to 8 carbons may be exemplified and specific examplesthereof are vinyl, allyl, 1-propenyl, butenyl, pentenyl, hexenyl,heptenyl, octenyl, cyclohexenyl and 2,6-octadienyl.

(iv) With regard to the lower alkynyl, linear or branched alkynyl having2 to 8 carbons may be exemplified and specific examples thereof areethynyl, propynyl, butynyl, pentynyl, hexynyl; heptynyl and octynyl.

(v) The halogen means each of fluorine, chlorine, bromine and iodineatoms.

(vi) With regard to the aryl and a group wherein one hydrogen atom isremoved from the aromatic ring formed together with two carbon atomsbeing adjacent to each, monocyclic, bicyclic or tricyclic aryl having 6to 14 carbons may be exemplified and specific examples thereof arephenyl, naphthyl, indenyl and anthranyl.

(vii) The alkylene moiety of the aralkyl and the heterocyclic alkyl hasthe same meaning as that where one hydrogen atom is removed from thedefinition for the above lower alkyl (i).

(viii) With regard to the aryl moiety of the aralkyl, a bicyclic fusedring group wherein the above aryl is fused to cycloalkyl may beexemplified in addition to the groups exemplified in the definition forthe above aryl (vi) and specific examples thereof are indanyl,1,2,3,4-tetrahydronaphthyl and 6,7,8,9-tetrahydro-5H-benzocycloheptyl.

(ix) With regard to the heterocyclic group, the heterocyclic moiety ofthe heterocyclic alkyl and a group wherein one hydrogen atom is removedfrom the heterocyclic ring formed together with two carbon atoms beingadjacent to each, a five- or six-membered monocyclic heterocyclic groupcontaining at least one atom selected from a nitrogen atom, an oxygenatom and a sulfur atom and a fused ring heterocyclic group which isbicyclic or tricyclic where three- to eight-membered rings are fused andwhich contains at least one atom selected from a nitrogen atom, anoxygen atom and a sulfur atom may be exemplified and specific examplesthereof are pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,benzimidazolyl, 2-oxobenzimidazolyl, benzotriazolyl, benzofuryl,benzothienyl, purinyl, benzoxazolyl, benzothiazolyl, benzodioxolyl,indazolyl, indolyl, isoindolyl, quinolyl, isoquinolyl, phthalazinyl,naphthylidinyl, quinoxalinyl, pyrrolyl, pyrazolyl, quinazolinyl,cinnolinyl, triazolyl, tetrazolyl, imidazolyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, thienyl, furyl, pyrrolidinyl,2,5-dioxopyrrolidinyl, thiazolidinyl, oxazolidinyl, piperidyl,piperidino, piperazinyl, homopiperazinyl, homopiperidyl, homopiperidino,morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, pyranyl,tetrahydropyridyl, tetrahydropyranyl, tetrahydrofuranyl,tetrahydroquinolyl, tetrahydroisoquinolyl, octahydroquinolyl andindolinyl.

(x) With regard to the heterocyclic group formed together with theadjacent nitrogen atom, a five- to six-membered monocyclic heterocyclicgroup containing at least one nitrogen atom (said monocyclicheterocyclic group may contain other nitrogen atom, oxygen atom orsulfur atom) and a fused ring heterocyclic group which is bicyclic ortricyclic where three- to eight-membered rings are fused and whichcontains at least one nitrogen atom (said fused ring heterocyclic groupmay contain other nitrogen atom, oxygen atom or sulfur atom) may beexemplified and specific examples thereof are pyridyl,tetrahydropyridyl, indolinyl, isoindolinyl, pyrrolidinyl, thiazolinyl,oxazolidinyl, piperidino, homopiperidino, piperazinyl, homopiperazinyl,morpholino, thiomorpholino, perhydroazepinyl, perhydroazocinyl,tetrahydroquinolyl, tetrahydroisoquinolyl, octahydroquinolyl,benzimidazolyl, indazolyl, indolyl, isoindolyl, purinyl, dihydroindolyl,pyrrolyl, dihydropyrrolyl, pyrazolyl, triazolyl, tetrazolyl andimidazolyl.

(xi) With regard to the substituent in the substituted lower alkyl andthe substituted lower alkanoyl, the number of the substituent(s), whichmay be the same or different; is 1 to 3 and examples thereof are thefollowing. Thus cycloalkyl, lower alkanoyl, lower alkoxy, aryloxy,substituted aryloxy [with regard to the substituent in said substitutedaryloxy, the number of the substituent(s), which may be the same ordifferent, is 1 to 3 and examples thereof are lower alkyl, lower alkoxy,lower alkoxycarbonyl, halogen, cyano, nitro, hydroxy, carboxy and amino;here, the lower alkyl has the same meaning as the above-mentioned loweralkyl (i), the halogen has the same meaning as the above-mentionedhalogen (v) and the lower alkyl moiety in the lower alkoxy and the loweralkoxycarbonyl has the same meaning as the above-mentioned lower alkyl(i)], aralkyloxy, substituted aralkyloxy [with regard to the substituentin said substituted aralkyloxy, the number of the substituent(s), whichmay be the same or different, is 1 to 3 and examples thereof are loweralkyl, lower alkoxy, lower alkoxycarbonyl, halogen, cyano, nitro,hydroxy, carboxy and amino; here, the lower alkyl has the same meaningas the above-mentioned lower alkyl (i), the halogen has the same meaningas the above-mentioned halogen (v) and the lower alkyl moiety in thelower alkoxy and the lower alkoxycarbonyl has the same meaning as theabove-mentioned lower alkyl (i)], lower alkanoyoxy, loweralkoxycarbonyl, halogen, cyano, nitro, hydroxy, carboxy, amino, loweralkylthio, substituted lower alkyl [with regard to the substituent insaid substituted lower alkyl, the number of the substituent(s) which maybe the same or different, is 1 to 3 and examples thereof are hydroxy andhalogen], the substituted lower alkanoyl [with regard to the substituentin the substituted lower alkanoyl, the number of the substituent(s),which may be the same or different, is 1 to 3 and an example thereof ishalogen], mono- or di-lower alkylamino, lower alkylsulfonyl, loweralkylsulfinyl, lower alkoxycarbonylamino, lower alkanoylamino, mono- ordi-lower alkylaminocarbonyl, mono- or di-lower alkylaminocarbonyloxy, aheterocyclic group and the like are exemplified.

The aryl moiety in the aryloxy and the aralkyloxy, the cycloalkyl, thehalogen, the heterocyclic group and the lower alkyl moiety in the loweralkanoyl, the lower alkoxy, the lower alkanoyloxy, the loweralkoxycarbonyl, the lower alkylthio, the lower alkylsulfonyl, the loweralkylsulfinyl, the lower alkoxycarbonylamino and the lower alkanoylaminoshown here have the same meanings as the above-mentioned aryl (vi),cycloalkyl (ii), halogen (v), heterocyclic group (ix) and lower alkyl(i), respectively. The alkylene moiety in the aralkyloxy has the samemeaning as that where one hydrogen atom is removed from theabove-mentioned lower alkyl (i).

The lower alkyl moiety of the mono- or di-lower alkylamino, the mono- ordi-lower alkylaminocarbonyl and the mono- or di-loweralkylaminocarbonyloxy each has the same meaning as the above-mentionedlower alkyl (i). Two lower alkyl moieties in the di-lower alkylamino,the di-lower alkylaminocarbonyl and the di-lower alkylaminocarbonyloxyeach may be the same or different.

(xii) With regard to the substituent in the substituted aryl, thesubstituted aralkyl, the substituted cycloalkyl, the substituted loweralkenyl, the substituted lower alkynyl, the substituted heterocyclicgroup, the substituted heterocyclic alkyl, the substituted heterocyclicgroup formed together with the adjacent nitrogen atom, the substitutedaromatic ring formed together with two carbon atoms being adjacent toeach and the substituted heterocycle formed together with two carbonatoms being adjacent to each, in addition to the above-mentioned groupsexemplified in the definition for the substituent (xi) in thesubstituted lower alkyl are lower alkyl, aryl, substituted aryl,aralkyl, substituted aralkyl, a heterocyclic group, a substitutedheterocyclic group, heterocyclic alkyl, substituted heterocyclic alkyland the like are exemplified. Further, with regard to the substituent inthe substituted aryl and the substituted-heterocyclic-group formedtogether with the adjacent nitrogen atom, it may be lower alkyl [saidlower alkyl has the same meaning as the above-mentioned lower alkyl (i)]or substituted lower alkyl [said lower alkyl has the same meaning as theabove-mentioned lower alkyl (i), and the number of the substituent(s),which may be the same or different, is 1 to 3 and examples thereof arehalogen, hydroxy, carboxy and lower alkoxycarbonyl; here, the halogenhas the same meaning as the above-mentioned halogen (v) and the loweralkyl moiety of the lower alkoxycarbonyl has the same meaning as theabove-mentioned lower alkyl (i)].

The lower alkyl, the aryl, the heterocyclic group moiety of theheterocyclic group and the heterocyclic alkyl, the alkylene moiety ofthe aralkyl and the heterocylic alkyl, and the aryl moiety of thearalkyl shown here have the same meanings as the above-mentioned loweralkyl (i), aryl (vi), heterocylic group (ix), alkylene moiety (vii) ofthe aralkyl and aryl moiety (viii) of the aralkyl, respectively. Withregard to the substituent in the substituted aryl, the substitutedaralkyl, the substituted heterocyclic group and the substitutedheterocyclic alkyl, the number of the substituent(s), which may be thesame or different, is 1 to 3 and examples thereof are lower alkyl [saidlower alkyl has the same meaning as the above-mentioned lower alkyl(i)], lower alkoxy [the lower alkyl moiety of said lower alkoxy has thesame meaning as the above-mentioned lower alkyl (i)] and halogen [saidhalogen has the same meaning as the above-mentioned halogen (v)].

With regard to the quaternary ammonium salt of compound (I), there is noparticular limitation so far as it is a quaternary ammonium salt whereinhalogenated lower alkyl (said lower alkyl and said halogen have the samemeanings as the above-mentioned ones, respectively), halogenated aralkyl(said halogen and said aralkyl have the same meanings as theabove-mentioned ones, respectively), hydroxy lower alkyl (said loweralkyl has the same meaning as the above-mentioned one) or the like isadded to a basic moiety of compound (I) and examples thereof are aquaternary ammonium salt prepared by the reaction of compound (I) havinga dimethylamino group with methyl iodide, a quaternary ammonium saltprepared by the reaction of compound (I) having a piperidino group withmethyl iodide, a quaternary ammonium compound prepared by the reactionof compound (I) having a pyrrolidino group with methyl iodide, aquaternary ammonium salt prepared by compound (I) having a morpholinogroup with benzyl bromide and a quaternary ammonium salt prepared byexchange of an iodide ion with a hydroxide ion in a quaternary ammoniumsalt prepared by the reaction of compound (I) having a pyrrolidino groupwith ethyl iodide.

With regard to the pharmaceutically acceptable salt of compound (I),that which has no toxicity and is soluble in water is preferred andexamples thereof are acid addition salts such as inorganic saltsincluding hydrochloride, hydrobromide, nitrate, sulfate, phosphate, etc.and organic salts including benzenesulfonate, benzoate, citrate,fumarate, gluconate, lactate, maleate, malate, oxalate,methanesulfonate, tartrate, etc., alkaline metal salts such as sodiumsalt and potassium salt etc., alkaline earth metal salt such asmagnesium salt and calcium salt etc., metal salts such as aluminum saltand zinc salt, ammonium salt such as ammonium and tetramethylammoniumetc., organic amine addition salts such as morpholine addition salt andpiperidine addition salt etc. and amino acid addition salt such asglycine addition salt, phenylalanine addition salt, lysine additionsalt, aspartic acid addition salt and glutamic acid addition salt etc.

Process for the production of compound (I) will be illustrated ashereunder.

In the process for the production as mentioned below, when the definedgroup changes under a reaction condition or when it is not appropriatefor conducting the process, the production may be easily carried out bysubjecting to a method commonly used in synthetic organic chemistry suchas by means of protection and deprotection of functional group [e.g.,Protective Groups in Organic Synthesis, the third edition, by T. W.Greene and Peter G. M. Wuts, John Wiley & Sons, Inc. (1999)]. Ifnecessary, order of reaction steps such as introduction of substituentsmay be changed.

Compound (I-a) may be prepared by the production process as shown below.

(wherein R², R³, R⁴, R⁵, R⁶, X and Y each have the same meaning asdefined above, respectively; R⁹ represents lower alkyl, allyl or benzyl;R¹⁰ and R¹¹ are the same or different and each represents lower alkyl orcycloalkyl, or R¹⁰ and R¹¹ may form a heterocyclic group together withthe adjacent nitrogen atom; and U represents halogen, alkoxysulfonyloxy,aryloxysulfonyloxy, alkylsulfonyloxy or arylsulfonyloxy.)

In the above definitions, the lower alkyl, the cycloalkyl and thehalogen each have the same meaning as that mentioned above,respectively. The alkyl moiety of the alkoxysulfonyloxy and thealkylsulfonyloxy and the aryl moiety of the aryloxysulfonyloxy and thearylsulfonyloxy each have the same as the above-mentioned lower alkyland aryl, respectively. The heterocyclic group formed together with theadjacent nitrogen atom has the same meaning as the above-mentioned one.

<Step 1>

Compound (IIIa) is used as a starting material and is made to react withfrom one equivalent to a large excess of YH (wherein Y has the samemeaning as defined above) by a process disclosed in the JP-A-7-61983 togive compound (IV). Incidentally, compound (IIIa) is able to besynthesized by the process disclosed in the JP-A-7-61983 or a processsimilar thereto.

<Step 2>

Compound (IV) is made to react with one equivalent to a large excess ofR⁵R⁶NH (wherein R⁵, and R⁶ each have the same meaning as defined above,respectively) or a hydrochloride thereof in an inert solvent in thepresence of one equivalent to a large excess of aqueous formaldehydesolution to give compound (I-a). It is also possible to use a substanceequivalent to formaldehyde such as trioxymethylene and paraformaldehydeinstead of an aqueous formaldehyde solution.

Since the reaction usually well proceeds under an acidic, condition, itis preferred to add an acid such as hydrochloric acid, acetic acid ortrifluoroacetic acid to the reaction system if necessary. The reactionis usually carried out at the temperature between 0° C. and the boilingpoint of the solvent used for the reaction, preferably, from roomtemperature to 80° C. and finishes within from 5 minutes to 100 hours.With regard to the inert solvent, water, methanol, ethanol, acetic acid,trifluoroacetic acid, dichloroethane, chloroform, tetrahydrofuran,dimethylacetamide, dimethylformamide, acetone or the like may be usedeither solely or as the mixture thereof. Preferably, a mixed solvent ofchloroform and acetic acid is used.

Compound (I-c) is produced from compound (I-b) by a process as shownbelow.

(wherein R², R³, R⁴, R⁵, R⁶, R^(5b), R^(6b), R⁸, U, n, X and Y each havethe same meaning as defined above, respectively.)

<Step 3>

Compound (I-b) is made to react with from one equivalent to a largeexcess of R⁸U (wherein R⁸ and U each have the same meaning as definedabove, respectively) in an inert solvent for 1 to 48 hour(s) at thetemperature of usually from −10° C. to the boiling point of the solventused for the reaction, preferably, at room temperature to give compound(I-c).

With regard to the inert solvent, it is possible to use, for example,water, methanol, ethanol, benzene, toluene, xylene, ethyl acetate,hexane, acetonitrile, dichloromethane, dichloroethane, chloroform,carbon tetrachloride, 1,4-dioxane, tetrahydrofuran, dimethylacetamide,dimethylformainide, acetone or the either solely or as the mixturethereof. Preferably, ethyl acetate, dichloroethane, chloroform, etc. areused.

Compound (I-b) is able to be produced from compound (I-c) by the processas shown below.

(wherein R³, R³, R⁴, R⁵, R⁶, R^(5b), R^(6b), U, n, X and Y each have thesame meaning as defined above, respectively.)

<Step 4>

Compound (I-c) is made to react with from one equivalent to a largeexcess of R⁵R⁶NH (wherein R⁵ and R⁶ each have the same meaning asdefined above, respectively) in an inert solvent for 1 to 100 hour(s) atthe temperature of usually from −10° C. to the boiling point of thesolvent-used for the reaction, preferably, at the temperature between20° C. and 100° C. to give compound (I-b).

With regard to the inert solvent, it is possible to use, for example,water, methanol, ethanol, benzene, toluene, xylene; ethyl acetate,hexane, acetonitrile, dichloromethane, dichloroethane, chloroform,carbon tetrachloride, 1,4-dioxane, tetrahydrofuran, dimethylacetamide,dimethylformamide, acetone or the like either solely or as the mixturethereof. Preferably, chloroform, dimethylformamide, etc. are used. Sincethe reaction usually well proceeds under a basic condition, it isdesirable to add an appropriate base to a reaction system if necessary.With regard to the base, triethylamine, diisopropylethylamine, pyridine,N-methylmorpholine, potassium carbonate, sodium hydride, potassiumhydride, calcium hydride, diisopropylethylamine,1,8-diazabicyclo[5.4.0]undec-7-ene, etc. may be used and, among them,triethylamine is preferred.

Compound (I-e) is able to be produced by the process mentioned belowusing compound (I-d) in compound (I-b).

(wherein R², R³, R⁴, n, X and Y each have the same meaning as definedabove, respectively; R¹⁴ and R¹⁵ are the same or different and eachrepresents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aralkyl or substituted or unsubstituted heterocyclicalkyl, or R¹⁴ and R¹⁵ may form a substituted or unsubstitutedheterocycle together with the adjacent CH(CH₂)_(m)N; R¹⁶ representshydrogen, substituted or unsubstituted lower alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted lower alkenyl,substituted or unsubstituted lower alkynyl, substituted or unsubstitutedaralkyl or substituted or unsubstituted aryl; and m represents aninteger of 0 to 3).

The lower alkyl, the cycloalkyl, the lower alkenyl, the lower alkynyl,the aralkyl, the heterocyclic alkyl and the aryl each have the samemeaning as defined above, respectively and the substituents thereof alsohave the same meanings as defined above, respectively.

With regard to the substituted or unsubstituted heterocycle formed fromR¹⁴ and R¹⁵ together with the adjacent CH(CH₂)_(m)N thereto,tetrahydropyridine, pyrrolidine, piperidine, homopiperidine, piperazine,homopiperazine, morpholine, thiomorpholine, perhydroazepine,perhydroadzocine, tetrahydroquinoline and tetrahydroisoquinoline, or thelike are exemplified and the substituent thereof has the same meaning asthe substituent for the above-mentioned heterocyclic group formedtogether with the adjacent nitrogen atom.

<Step 5>

When compound (I-d) is treated for 10 minutes to 24 hours, preferably, 1to 3 hour(s) in the presence of 2 to 4 equivalents of a reducing agentsuch as lithium aluminum hydride, diisopropyl lithium aluminum hydride,etc., preferably, diisopropyl lithium aluminum hydride usually at thetemperature from −78° C. to 40° C. in an inert solvent, compound (I-e)is able to be prepared.

With regard to the inert solvent, dichloromethane, chloroform, carbontetrachloride, dichloroethane, benzene, toluene, xylene,tetrahydrofuran, diethyl ether, etc. are used either solely or as themixture thereof and, preferably, dichloromethane or toluene is used.

Compound (I-f) is able to be produced from compound (I-d) by the processas mentioned below.

(wherein R², R³, R⁴, R¹⁴, R¹⁵, R¹⁶, n, X, Y and m each have the samemeaning as defined above, respectively.)

<Step 6>

When compound (I-d) is treated for 1 to 48 hours, preferably, 1 to 3hour(s) in the presence of from one equivalent to a large excess ofappropriate base at the temperature of usually from 0° C. to the boilingpoint of the solvent used for the reaction, preferably, at thetemperature between room temperature and 100° C. in an inert solvent,compound (I-f) is able to be produced.

With regard to the appropriate base, sodium hydroxide, lithiumhydroxide, potassium hydroxide, potassium carbonate, cesium carbonateand sodium methoxide are exemplified and sodium hydroxide is preferablyexemplified. With regard to the inert solvent, water, tetrahydrofuran,diethyl ether, methanol, ethanol, propanol, dichloromethane,dichloroethane, benzene, toluene, xylene, etc. may be used either solelyor as the mixture thereof and, preferably, tetrahydrofuran or methanolor a mixed solvent thereof with water is used.

Compound (I-h) is able to be produced by the following process fromcompound (I-g) in compound (I-c).

(wherein R², R³, R⁴, n, X, Y and m each have the same meaning as definedabove, respectively; R¹⁷ and R^(18a) each have the same meaning as theabove R¹⁴ and the above R¹⁵, respectively; and T represents alkalinemetal, ammonium, trialkylsilyl or trialkyltin.)

The alkyl in the trialkylsilyl and trialkyltin in the above definitionhas the same meaning as the above-mentioned lower alkyl. Examples of thealkaline metal are sodium, potassium or the like.

<Step 7>

When compound (I-g) is made to react with from one equivalent to a largeexcess, preferably, 2 to 4 equivalents of TN₃ (wherein T has the samemeaning as defined above) for 1 to 200 hours, preferably, for 3 to 48hours usually in the presence of from catalytic amount to a largeexcess, preferably, 0.5 to 2 equivalent(s) of appropriate additive foraccelerating the reaction at the temperature of from 0° C. to theboiling point of the solvent used for the reaction, preferably, at thetemperature between room temperature and 200° C. in an inert solvent,compound (I-h) is able to be produced.

Examples of the appropriate additive are silicon tetrachloride, lithiumchloride, aluminum chloride, ammonium chloride, trialkyltin chloride,dialkyltin oxide, trialkyl aluminum, triethylamine hydrochloride,triethylamine hydrobromide, sodium hydroxide, potassium tert-butoxide,sodium hydroxide, and zinc bromide and preferred examples are ammoniumchloride and dialkyltin oxide. With regard to the inert solvent, water,acetonitrile, dimethylformamide, dimethylacetamide,N-methyl-2-pyrrolidone, dimethyl sulfoxide, acetic acid, glacial aceticacid, tetrahydrofuran, benzene, toluene, xylene, etc. may be used eithersolely or as the mixture thereof. Preferably, dimethylformamide ortoluene is used.

Compound (I-i) is able to be produced by the following process fromcompound (I-c).

(wherein R², R³, R⁴, R⁵, R⁶, R⁸, U, n, X and Y each have the samemeaning as defined above, respectively; R¹⁸ represents substituted orunsubstituted lower alkyl; Q represents alkaline metal or alkaline earthmetal and, when Q represents alkaline metal, p represents 1, while, whenQ represents alkaline earth metal, p represents 2)

In the above definition, the alkaline metal has the same meaning as theabove-mentioned alkaline metal and examples of the alkaline earth metalare magnesium, calcium or the like.

<Step 8>

When compound (I-c) is made to react with from 1 equivalent to a largeexcess, preferably, 4 to 8 equivalents of (R¹⁸CO₂)_(p)Q (wherein R¹⁸, Qand p each have the same meaning as defined above, respectively) for 1to 100 hour(s), preferably, 3 to 72 hours at the temperature between 0°C. and the boiling point of the solvent used for the reaction,preferably, the temperature between 70° C. and 80° C. in an inertsolvent, compound (I-i) is able to be produced.

With regard to the inert solvent, dimethylacetamide,N-methyl-2-pyrrolidone, dimethyl sulfoxide, etc. may be used eithersolely or as the mixture thereof and preferably, dimethyl sulfoxide isused.

Compound (I-j) is able to be produced from compound (I-c) by the processas shown below.

(wherein R², R³, R⁴, R⁵, R⁶, R^(7a), R⁸, U, n, X and Y each have thesame meaning as defined above, respectively.)

<Step 9>

When compound (I-c) is made to react with from 1 equivalent to a largeexcess, preferably, 2 to 8 equivalents of R^(7a) SH (wherein R^(7a) hasthe same meaning as defined above) for 1 to 100 hour(s), preferably, 3to 72 hours in the presence of 1 equivalent to a great excess,preferably, 1 to 3 equivalent(s) of an appropriate base at thetemperature between 0° C. to the boiling point of a solvent used for thereaction, preferably, between 30° C. and 80° C. in an inert solvent,compound (I-j) is able to be produced.

With regard to the appropriate base, triethylamine,diisopropylethylamine, pyridine, N-methylmorpholine, potassiumcarbonate, sodium hydride, potassium hydride, calcium hydride,diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc. may beused and, among them, 1,8-diazabicyclo[5.4.0]undec-7-ene is preferred.With regard to the inert solvent, dichloromethane, chloroform, carbontetrachloride, dichloroethane, benzene, toluene, xylene, ethyl acetate,dimethylacetamide, N-methyl-2-pyrrolidone, dimethyl sulfoxide, etc. maybe used either solely or as the mixture thereof and, preferably,chloroform may be used.

Compound (I-1) is able to be produced by the process as mentioned belowfrom compound (I-k) in compound (I-j).

(wherein R², R³, R⁴, R¹⁵, R¹⁶, m, n, X and Y each have the same meaningas defined above, respectively.)

<Step 10>

Compound (I-l) is able to be produced from compound (I-k) by conductingthe reaction similar to the step 6 for Production Process 5.

Compound (I-m) is able to be produced from compound (I-i) by the processas mentioned below.

(wherein R², R³, R⁴, R¹⁸, n, X and Y each have the same meaning asdefined above, respectively.)

<Step 11>

Compound (I-m) is able to be produced from compound (I-i) by conductingthe reaction similar to the step 6 for Production Process 5.

Compound (I-n) is able to be produced from compound (I-m) by the processas mentioned below.

(wherein R², R³, R⁴, U, n, X and Y each have the same meaning as definedabove, respectively, and R^(7c) represents a group wherein hydrogen isremoved from the definition of the above R⁷.)

<Step 12>

When compound (I-m) is made to react with from one equivalent to a largeexcess, preferably, from 1 to 3 equivalent(s) of R^(7c)U (wherein R^(7c)and U each have the same meaning as defined above, respectively) for 1to 48 hour(s), preferably, 3 to 24 hour(s) in the presence of from 1equivalent to a large excess, preferably, 1 to 3 equivalent (s) of anappropriate base, at the temperature between 0° C. and the boiling pointof the solvent used for the reaction, preferably, at the temperaturebetween room temperature and 80° C. in an insert solvent, compound (I-n)is able to be prepared.

Examples of the appropriate base are potassium carbonate, sodiumhydride, potassium hydride, calcium hydride and lower alkyllithium andpreferred ones are sodium hydride, potassium hydride, etc. With regardto the inert solvent, dichloromethane, chloroform, carbon tetrachloride,dichloroethane, benzene, toluene, xylene, ethyl acetate,dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone,tetrahydrofuran, diethyl ether, etc. may be used either either solely oras the mixture thereof. Preferably, chloroform is used.

Compound (I-o) is able to be produced by the process as mentioned belowfrom compound (I-ma) which is compound (I-m) wherein n is 1.

(wherein R², R³, R⁴, X and Y each have the same meaning as definedabove, respectively.)

<Step 13>

When compound (I-ma) is treated for 1 to 48 hour(s), preferably, 3 to 24hours in the presence of from 1 equivalent to a large excess,preferably, 3 to 6 equivalents of an appropriate oxidizing agent at thetemperature between 0° C. and the boiling point of the solvent used forthe reaction, preferably, the temperature between room temperature and60° C. in an inert solvent, compound (I-o) is able to be prepared.

With regard to the appropriate oxidizing agent, manganese dioxide,chromic acid, pyridinium chlorochromate, pyridinium dichromate,potassium permanganate, sulfur trioxide-pyridine and oxone areexemplified and manganese dioxide is preferably exemplified. With regardto the inert solvent, dichloromethane, chloroform, carbon tetrachloride,dichloroethane, benzene, toluene, xylene, ethyl acetate, acetic acid,propionic acid, butyric acid, trifluoroacetic acid, water, pyridine,dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone,1,4-dioxane, tetrahydrofuran, diethyl ether, etc. may be used eithersolely or as the mixture thereof. Preferably, dimethylformamide,tetrahydrofuran, etc. may be used.

Compound (I-p) is able to be produced by the process as mentioned belowfrom compound (I-o).

(wherein R², R³, R⁴, X and Y each have the same meaning as definedabove, respectively.)

<Step 14>

When compound (I-o) is made to react with from 1 equivalent to a largeexcess, preferably, 1 to 3 equivalent(s) of hydroxylamine or itshydrochloride, sulfate, p-toluenesulfonate, etc. thereof,O-phenylcarbamylhydroxylamine or its hydrochloride, sulfate,p-toluenesulfonate, etc. thereof or N-hydroxybenzamide, preferably, withhydroxylamine for 1 to 48 hour (s), preferably, 3 to 24 hours at thetemperature between 0° C. and the boiling point of the solvent used forthe reaction, preferably, between room temperature and 90° C. in aninert solvent, compound (I-p) is able to be prepared. If necessary,addition of 1 equivalent to a large excess, preferably, 1 to 3equivalent(s) of an appropriate dehydrating agent, addition of 1equivalent to a large excess, preferably, 2 to 6 equivalents of anappropriate base or irradiation with microwave may be carried out.

With regard to the appropriate dehydrating agent, acetic anhydride,phthalic anhydride, sodium hydrogen sulfate, oxone, sodium formate,dialkyltin oxide, alumina, silica gel, sodium acetate, formamide,diphosphorus pentaoxide, ferric chloride, formic acid, acetic acid,propionic acid, phosphorus oxychloride, p-toluenesulfonic acid, etc. maybe exemplified and, preferably, acetic anhydride, phthalic anhydride,etc. may be exemplified. With regard to the appropriate base,triethylamine, pyridine, sodium hydride, potassium hydride, etc. may beexemplified and, preferably, triethylamine or pyridine may beexemplified.

With regard to the inert solvent, dichloromethane, chloroform, carbontetrachloride, dichloroethane, benzene, toluene, xylene, nitrobenzene,acetonitrile, ethyl acetate, acetic acid, propionic acid, butyric acid,trifluoroacetic acid, pyridine, dimethylformamide, dimethylacetamide,N-methyl-2-pyrrolidone, 1,4-dioxane, tetrahydrofuran, diethyl ether,methanol, ethanol, propanol, etc. may be used either solely or as themixture thereof. Preferably, acetonitrile, dimethylformamide, etc. maybe used.

Compound (I-q) is able to be produced by the process as mentioned belowfrom compound (I-p).

(wherein R², R³, R⁴, T, X and Y each have the same meaning as defineabove, respectively.)

<Step 15>

Compound (I-q) is able to be produced using compound (I-p) by conductingthe reaction similar to that in the step 7 of Producing Process 6.

Compound (I-r) is able to be produced by the process as mentioned belowfrom compound (I-c)

(wherein R², R³, R⁴, R^(5b), R^(6b), R⁸, U, n, X and Y each have thesame meaning as defined above, respectively; and Q^(a) represents thesame alkaline metal as defined above.)

<Step 16>

When compound (I-c) is made to react with from 1 equivalent to a largeexcess, preferably, 2 to 4 equivalents of Q^(a)CN (wherein Q^(a) has thesame meaning as defined above), preferably sodium cyanide for 1 to 48hour(s), preferably, 3 to 24 hours at the temperature between roomtemperature and the boiling point of the solvent used for the reaction,preferably, between 40° C. and 80° C. in an inert solvent, compound(I-r) is able to be produced.

With regard to the inert solvent, dichloromethane, chloroform, carbontetrachloride, dichloroethane, benzene, toluene, xylene,dimethylformamide, dimethylacetamide, N-methyl-2-pyrrolidone,1,4-dioxane, tetrahydrofuran, etc. may be used either solely or as themixture thereof. Preferably, dimethylformamide, etc. may be used.

Compound (I-s) is able to be produced by the process as shown below fromcompound (I-r).

(wherein R², R³, R⁴, T, n, X and Y each have the same meaning as definedabove, respectively.)

<Step 17>

Compound (I-s) is able to be produced by conducting the reaction similarto the step 7 of Producing Process 6 using compound (I-r).

Compound (I-t) is able to be produced by the process as mentioned-belowfrom compound (I-r).

(wherein R², R³, R⁴, n, X and Y each have the same meaning as definedabove, respectively.)

<Step 18>

Compound (I-t) is able to be produced by conducting the reaction similarto the step 6 of Producing Process 5 using compound (I-r).

Compound (I-u) is able to be produced by the process as mentioned belowfrom compound (IIIb).

(wherein R², R³, R⁴, R⁵, R⁶, R^(5b), R^(6b), R^(7c), R⁸, R⁹, R¹⁰, R¹¹,R¹⁸, Q, p, U, X and Y each have the same meaning as defined above,respectively.)

<Step 19>

Compound (V) is able to be produced by conducting the reaction similarto the step 8 of Producing Process 7 using compound (IIIb).

<Step 20>

Compound (VI) is able to be-produced by conducting the reaction similarto the step 6 of Producing Process 5 using compound (V).

<Step 21>

Compound (VII) is able to be produced by conducting the reaction similarto the step 13 of Producing Process 12 using compound (VI).

<Step 22>

When compound (VII) is treated for 10 minutes to 24 hours, preferably, 1to 4 hour(s) in the presence of 2 to 4 equivalent(s) of an oxidizingagent such as silver nitrate, silver (I) oxide, silver (II) oxide,chromic acid, pyridinium chlorochromate, pyridinium dichlorochromate,potassium permanganate, sodium periodate, sodium perchlorate,hydrogen-peroxide and sodium chlorite, preferably, silver nitrate orsodium perchlorate usually at the temperature between 0° C. and 80° C.in an inert solvent, compound (VIII) is able to be produced. Ifnecessary, 0.1 to 4 equivalent(s) of an organic substance such as aceticacid or an inorganic substance such, as sulfuric acid, sodium dihydrogenphosphate, sulfamic acid and ruthenium oxide may be added as anadditive.

With regard to the inert solvent, diethyl ether, tetrahydrofuran,1,4-dioxane, dimethylformamide, dimethylacetamide, dimethyl sulfoxide,benzene, toluene, xylene, dichloromethane, chloroform,1,2-dichloroethane, acetonitrile, ethyl acetate, methyl acetate, methylethyl ketone, hydrochloric acid, acetic acid, acetic anhydride, sulfuricacid, water, etc. may be exemplified and, preferably, acetonitrile,water, etc. are exemplified. Each of them may be used solely or as themixture thereof.

<Step 23>

When compound (VIII) is made to react with 1 to 20 equivalent(s) ofhalogenating agent for 10 minutes to 24 hours usually at the temperaturebetween 0° C. and 80° C., preferably, at room temperature in an inertsolvent and, after that, it is made to react with from 1 equivalent to alarge excess of R^(7c)OH (wherein R^(7c) has the same meaning as definedabove), compound (IX) is able to be produced.

With regard to the halogenating agent, thionyl chloride, oxalyl chlorideand phosphorus oxychloride are exemplified and, preferably, thionylchloride is exemplified. With regard to the inert solvent,dichloromethane, chloroform, tetrahydrofuran, dimethylformamide,dimethylacetamide, 1,4-dioxane, acetonitrile, benzene, toluene andxylene are exemplified and each of them may be used either solely or asthe mixture thereof. With regard to the inert solvent, dichloromethaneis preferably exemplified.

<Step 24>

Compound (X) is able to be produced by conducting the reaction similarto the step 2 of Producing Process 1 using compound (IX).

<Step 25>

Compound (XI) is able to be produced by conducting the reaction similarto the step 3 of Producing Process 2 using compound (X).

<Step 26>

Compound (I-u) is able to be produced by conducting the reaction similarto the step 1 of Producing Process 1 using compound (XI).

Compound (I-v) is able to be produced by the process as shown below fromcompound (I-u).

(wherein R², R³, R⁴, R^(7c), X and Y each have the same meaning asdefined above, respectively.)

<Step 27>

Compound (I-v) is able to be produced by conducting the reaction similarto the step 6 of Producing-Process-5 using compound (I-u).

Compound (I-w) is able to be produced by the process as shown below fromcompound (I-v).

(wherein R², R³, R⁴, R^(5a), R^(6a), X and Y each have the same meaningas defined above, respectively.)

<Step 28>

When compound (I-v) is made to react with 1 to 20 equivalent(s) of ahalogenating agent for 10 minutes to 24 hours usually at the temperaturebetween 0° C. and 80° C., preferably, at room temperature in an inertsolvent and, after that, made to react with 1 equivalent to a largeexcess of R^(5a)R^(6a)NH (wherein R^(5a) and R^(6a) each have the samemeaning as defined above, respectively), compound (I-w) is able to beproduced. If necessary, 1 equivalent to a large excess of an appropriatebase may be added thereto.

With regard to the halogenating agent, thionyl chloride, oxalylchloride, phosphorus oxychloride, etc. may be exemplified and,preferably, thionyl chloride is exemplified. With regard to theappropriate base, pyridine, triethylamine, diisopropylethylamine,N-methylmorpholine, etc. may be exemplified and, preferably,triethylamine may be exemplified. With regard to the inert solvent,dichloromethane, chloroform, tetrahydrofuran, dimethylformamide,dimethylacetamide, 1,4-dioxane, acetonitrile, benzene, toluene, xylene,etc. may be exemplified and each of them may be used solely or as themixture thereof. With regard to the inert solvent, dichloromethane ispreferably exemplified.

In the production of compound (I-w), it is also possible to apply themethod which has been-commonly used in peptide chemistry. Thus, whencompound (I-v) is made to react with 1 to 10 equivalent(s) ofR^(5a)R^(6a)NH (wherein R^(5a) and R^(6a) each have the same meaning asdefined above, respectively) together with 0.5 to 10 equivalent(s) of anappropriate condensing agent usually at the temperature between 0° C.and 50° C. for 10 minutes to 70 hours in an inert solvent, compound(I-w) is able to be produced.

With regard to the inert solvent, diethyl ether, tetrahydrofuran,1,4-dioxane, dimethylformamide, dimethylacetamide, dimethyl sulfoxide,benzene, toluene, xylene, acetonitrile, ethyl acetate, pyridine,dichloromethane, chloroform and carbon tetrachloride may be exemplifiedand, preferably, tetrahydrofuran and dimethylformamide may beexemplified.

With regard to the appropriate condensing agent,1,3-dicyclohexylocarbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-bonded polystyrene resin(EDC resin), etc. may be exemplified. It is also possible to add anadditive such as N-hydroxysuccinimide,3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, 1-hydroxybenzotriazole,preferably, 1-hydroxybenzotriazole thereto.

EDC resin is able to be produced by a process mentioned in TetrahedronLetters, volume 34, no. 48, page 7685 (1993).

Compound (I-y) is able to be produced by the process shown below fromcompound (I-x) in compound (I).

(wherein R¹, R³, R⁴, n, X and Y each have the same meaning as definedabove, respectively; and R²² and R²³ are the same or different and eachrepresents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aralkyl or substituted or unsubstituted heterocyclicalkyl.)

In the above definitions, the lower alkyl, the cycloalkyl, the loweralkenyl, the lower alkynyl, the aralkyl and the heterocyclic alkyl eachhave the same meaning as defined above, respectively and the substituentthereof also has the same meaning as defined above, respectively.

<Step 29>

When compound (I-x) is made to react with 1 equivalent to a largeexcess, preferably, 1 to 10 equivalent(s) of R²²R²³CO (wherein R²² andR²³ each have the same meaning as defined above, respectively) usuallyat the temperature between −78° C. and 100° C., preferably, thetemperature between 0° C. and 50° C. in the presence of 1 equivalent tolarge excess, preferably, 1 to 3 equivalent(s) of an appropriatereducing agent, for 10 minutes to 48 hours in an inert solvent, compound(I-y) is able to be produced.

With regard to the appropriate reducing agent, sodium borohydride,sodium triacetoxyborohydride, sodium cyanoborohydride, etc. may beexemplified and, preferably, sodium cyanoborohydride may be exemplified.If necessary, catalytic amount to solvent amount, preferably, 0.5equivalent to solvent amount of appropriate acid may be added thereto.With regard to the appropriate acid, formic acid, acetic acid,trifluoroacetic acid, propionic acid, hydrochloric acid, etc. may beexemplified and, preferably, acetic acid may be exemplified.

With regard to the inert solvent, dichloromethane, chloroform, carbontetrachloride, dichloroethane, benzene, toluene, xylene, diethyl ether,1,4-dioxane, dimethylformamide, dimethylacetamide, acetonitrile, hexane,formic acid, acetic acid, trifluoroacetic acid, propionic acid,hydrochloric acid, etc. may be exemplified and each of them may be usedsolely or as the mixture thereof. Preferably, tetrahydrofuran, aceticacid, etc. may be exemplified.

Transformation of each functional group in compound (I) and the startingcompounds and transformation of a functional group contained in thesubstituent may also be carried out a known method [such as a methodmentioned in Comprehensive Organic Transformation, second edition, by R.C. Larock, John Wiley & Sons, Inc. (1999)], etc.

When the above-mentioned method, etc. are carried out in anappropriately combined manner, it is possible to prepare compound (I)having a desired functional group at a desired position.

Isolation and purification of an intermediate and the product in theabove producing process may be carried out by an appropriate combinationof the methods which are used in common organic synthesis such asfiltration, extraction, washing, drying, concentration, crystallization,various kinds of chromatographies, etc. It is also possible to conduct apurifying method commonly used in general parallel synthetic methodssuch as purifying methods using scavenger resin and ion-exchange resin.It is also possible that the intermediate is not particularly purifiedbut just subjected to the next reaction.

In some of compound (I), there may be isomers such as regioisomer,geometric isomer, optical isomer or tautomer and all possible isomersincluding the above and mixtures of any ratio of said isomers can beused as an agent for the prevention and/or treatment of asthma.

When a salt of compound (I) is to be prepared, the product may be justpurified in case a salt of compound (I) is obtained while, in casecompound (I) is obtained in a free form, compound (I) may be dissolvedor suspended in an appropriate solvent and isolated and purified afteraddition of acid or base.

Some of compound (I) or the pharmaceutically acceptable salt thereof maybe present in a form of an adduct with water or with various solvents,and such adducts are also able to be used as an agent for preventionand/or treatment of asthma of the present invention.

Although specific examples of compound (I) which is able to be used asan agent for prevention and/or treatment of asthma of the presentinvention are shown in the following Tables 1 to 13, the scope of thecompound used as an agent for prevention and/or treatment of asthma ofthe present invention is not limited to thereto. TABLE 1

Compound No.  

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

TABLE 2

Compound No.  

 

Mass Spectrometric Data 28

MS m/z 438 (M + H)⁺ 29

MS m/z 421 (M + H)⁺ 30

MS m/z 409 (M + H)⁺ 31

MS m/z 451 (M + H)⁺ 32

MS m/z 506 (M + H)⁺ 33

MS m/z 513 (M + H)⁺ 34

MS m/z 514 (M + H)⁺ 35

MS m/z 496 (M + H)⁺ 36

MS m/z 425 (M + H)⁺ 37

MS m/z 427 (M + H)⁺ 38

MS m/z 425 (M + H)⁺ 39

MS m/z 471 (M + H)⁺

TABLE 3

Compound No.  

 

Mass Spectrometric Data 40

MS m/z 514 (M + H)⁺ 41

MS m/z 497 (M + H)⁺ 42

MS m/z 485 (M + H)⁺ 43

MS m/z 527 (M + H)⁺ 44

MS m/z 582 (M + H)⁺ 45

MS m/z 589 (M + H)⁺ 46

MS m/z 590 (M + H)⁺ 47

MS m/z 572 (M + H)⁺ 48

MS m/z 501 (M + H)⁺ 49

MS m/z 503 (M + H)⁺ 50

MS m/z 501 (M + H)⁺ 51

MS m/z 547 (M + H)⁺

TABLE 4

Compound No.  

 

Mass Spectrometric Data 52

MS m/z 452 (M + H)⁺ 53

MS m/z 435 (M + H)⁺ 54

MS m/z 423 (M + H)⁺ 55

MS m/z 465 (M + H)⁺ 56

MS m/z 520 (M + H)⁺ 57

MS m/z 527 (M + H)⁺ 58

MS m/z 528 (M + H)⁺ 59

MS m/z 510 (M + H)⁺ 60

MS m/z 439 (M + H)⁺ 61

MS m/z 441 (M + H)⁺ 62

MS m/z 439 (M + H)⁺ 63

MS m/z 485 (M + H)⁺

TABLE 5

Compound No.  

 

Mass Spectrometric Data 64

MS m/z 466 (M + H)⁺ 65

MS m/z 449 (M + H)⁺ 66

MS m/z 437 (M + H)⁺ 67

MS m/z 479 (M + H)⁺ 68

MS m/z 534 (M + H)⁺ 69

MS m/z 541 (M + H)⁺ 70

MS m/z 542 (M + H)⁺ 71

MS m/z 524 (M + H)⁺ 72

MS m/z 453 (M + H)⁺ 73

MS m/z 455 (M + H)⁺ 74

MS m/z 453 (M + H)⁺ 75

MS m/z 499 (M + H)⁺

TABLE 6

Compound No.  

 

mass Spectrometric Data 76

MS m/z 466 (M + H)⁺ 77

MS m/z 449 (M + H)⁺ 78

MS m/z 437 (M + H)⁺ 79

MS m/z 479 (M + H)⁺ 80

MS m/z 534 (M + H)⁺ 81

MS m/z 541 (M + H)⁺ 82

MS m/z 542 (M + H)⁺ 83

MS m/z 524 (M + H)⁺ 84

MS m/z 453 (M + H)⁺ 85

MS m/z 455 (M + H)⁺ 86

MS m/z 453 (M + H)⁺ 87

MS m/z 499 (M + H)⁺

TABLE 7

Compound No.  

 

88

89

90

TABLE 8

Compound No.  

92

93

94

95

96

97

98

99

100

101

102

TABLE 9

Compound No.  

103

104

105

106

107

108

109

110

111

TABLE 10

Compound No.  

112

113

114

115

116

117

118

119

TABLE 11

Compound No.

120

121

122

123

TABLE 12

Compound No.

124

125

TABLE 14 Compound No. Analytical Data 5 MS m/z 508(M + H)⁺ 6 MS m/z563(M + H)⁺ 7 MS m/z 570(M + H)⁺ 8 MS m/z 571(M + H)⁺ 9 MS m/z 553(M +H)⁺ 10 MS m/z 484(M + H)⁺ 11 MS m/z 482(M + H)⁺ 12 MS m/z 528(M + H)⁺

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a suppressive effect of compound 1 (by intraperitonealadministration) on bronchoconstriction induced by antigen. In FIG. 1,each of the symbols (##, **) has the following meaning, respectively.

##: p=0.0043 (ratio of a positive control group to a negative controlgroup; Student's t-test)

**: p=0.0047 (ratio of a group to which compound 1 was intraperitoneallyadministered to a positive control group; Student's t-test)

FIG. 2 shows a suppressive effect of compound 1 (by oral administration)on bronchoconstriction induced by antigen. In FIG. 2, each of thesymbols (###, *) has the following meaning, respectively.

###: p<0.0001 (ratio of a positive control group to a negative controlgroup; Student's t-test)

*: p=0.0248 (ratio of a group to which compound 1 was orallyadministered to a positive control group; Student's t-test)

FIG. 3 shows a suppressive effect of compound 1 on airwayhyperreactivity induced by antigen. In FIG. 3, the symbol (#) has thefollowing meaning.

#: p=0.0182 (ratio of a positive control group to a negative controlgroup; Student's t-test)

FIG. 4 shows a suppressive effect of compound 1 on eosinophilinfiltration in airway induced by antigen. In FIG. 4, each of thesymbols (###, ***) has the following meaning, respectively.

###: p=0.0009 (ratio of a positive control group to a negative controlgroup; Aspin-Welch test)

***: p=0.0030 (ratio of a group to which compound 1 was administered toa positive control group; Student's t-test)

****: p=0.0015 (ratio of a reference group to a positive control group;Student's t-test)

Pharmacological action of the compounds is illustrated by way of TestExamples.

TEST EXAMPLE 1 Antagonistic Action on GPR4

Cells for GPR4 assay prepared in Referential Example 61 (said assaycells express GPR4 by stimulation with 17β-estradiol) were seeded onwhite plates at the rate of 10⁵ cells/well, and a solution where17β-estradiol (manufactured by Sigma) was diluted with a medium so as tomake 10 nmol/L in the reaction solution and test compounds were added tothe plate and the mixture was incubated at 37° C. for 6 hours in a 5%CO₂ incubator. After that, a Steady Glo Luciferase Assay System(manufactured by Promega) solution was added to stop the reaction andthe amount of luminescence during one second was measured using a TopCount (Packard, Meriden, Conn., U.S.

Activity of the each of test compounds (antagonistic action) wasexpressed by an inhibition rate calculated on the basis of counts persecond in the presence or absence of 17β-estradiol as shown by thefollowing formula. IC₅₀ value was calculated from the inhibition rate bya linear approximate analysis of the Logit-Log conversion method.

In the formula, A, B and C each have the following meaning,respectively.

A: counts per second when 17β-estradiol and the test compound were added

B: counts per second when neither 17β-estradiol nor the test compoundwas added

C: counts per second when only 17β-estradiol was added

Inhibition Rate (%) [1−{(A−B)/(C−B)}]×100

The result is shown in Table 15. TABLE 15 Compound Nos. IC₅₀ (nmol/L) 14.0 2 3.2 3 2.3 4 5.8 5 14

From the above results, compound (I) has been shown to have anantagonistic action to GPR4.

TEST EXAMPLE 2 Suppressive Effect on Antigen-InducedBronchoconstruction, Airway Hyperreactivity and Eosinophil Infiltrationin Airway

Suspention prepared by mixing of 50 μg of ovalbumin and 1 mg of aluminumhydroxide in a physiological saline solution (Otsuka PhysiologicalSaline; manufactured by Otsuka. Pharmaceutical) was intraperitoneallyadministered to BALB/c male mice (7 weeks age) for two times with aninterval of one week to sensitize and, after 14, 18 and 22 days from thefinal sensitization, 1% solution of ovalbumin in a physiological salinesolution or a physiological saline solution (a negative control group)was inhaled for 30 minutes respectively to induce an antigen-antibodyreaction. In the case of measurement of bronchoconstriction, compound 1was suspended in a 0.5% aqueous solution of methylcellulose (solvent)and, 1 hour before inhalation of antigen after 14 days from the finalsensitization 100 mg/kg of the suspention was orally administered or, 5minutes before that, 30 mg/kg was intraperitoneally administered. In thecase of measurement of airway hyperreactivity or eosinophil infiltrationin airway, compound 1 was suspended as mentioned above and, after 14, 18and 22 days from the final sensitization, 100 mg/kg of the suspentionwas orally administered 1 hour before and 6 hours after inhalation ofeach antigen. As a reference drug, prednisolone which is a steroid usedfor the treatment of asthma was orally administered at a dose of 30mg/kg (as a solution being suspended in 0.5% aqueous solution ofmethylcellulose) once, 1 hour before inhalation of each antigen after14, 18 and 22 days from the final sensitization. In a positive controlgroup, the solvent was administered instead of a suspension of a testcompound. (In the case of measurement of bronchoconstriction, thesolvent was administered once, 1 hour before inhalation of antigen after14 days from the final sensitization as for oral administration, or thesolvent was administered once 30 minutes before inhalation of antigenafter 14 days from the final sensitization as for intraperitonealadministration. In the case of measurement of airway hyperreactivity andeosinophil infiltration in airway, the solvent was administered 1 hourbefore and 6 hours after inhalation of each antigen after 14, 18 and 22days from the final sensitization.)

With regard to the bronchoconstriction, airway resistance (penh) wasevaluated by a measuring apparatus for respiration function of mice(BioSystems XA; Buxco Electronics, Inc., Sharon, Conn., U.S.A.) for 30minutes from immediately after inhalation of antigen after 14 days fromthe final sensitization and evaluation was conducted by calculating thearea under curve during the 30 minutes (AUC₀₋₃₀ min). Further, after 24hours from final inhalation of antigen, airway hyperreactivity andeosinophil infiltration in bronchoalveolar lavage fluid were evaluated.

With regard to an airway hyperreactivity test, bronchoconstriction afterinhalation of 1.5 to 25 mg/ml of methacholine for 3 minutes (inhaledafter 24 hours from 22 days after the final sensitization) wasdetermined by a measuring apparatus for respiration function of mice(BioSystems XA; Buxco Electronics, Inc., Sharon, Conn., U.S.A.) andevaluation was conducted by calculating the area under curve (AUC) froma curve of dose of methacholine vs. bronchoconstriction. With regard toeosinophil filtration, total cell numbers in the bronchoalveolar lavagefluid were measured by an automatic measuring apparatus for blood cellcount (Celltac α MEK-6158; Nippon Koden, Tokyo), then the smears wereprepared by Cytospin 3 (Shandon, Inc., Pittsburgh, Pa., U.S.A.) andevaluation was conducted by a morphological classification under amicroscope. Eosinophil numbers were calculated by multiplying the totalcell numbers by percentage of eosinophil cells. The test was conductedfor a group comprising ten mice.

Result for bronchoconstriction is shown in FIG. 1 (by intraperitonealadministration) and in FIG. 2 (by oral administration), result forairway hyperreactivity is shown in FIG. 3 and result for eosinophilinfiltration in airway is shown in FIG. 4.

As shown in FIG. 1, AUC_(0-30min) of the bronchoconstriction in thepositive control group (18.22±1.02; mean value±standard error)significantly increased as compared with AUC_(0-30min) of the negativecontrol group (14.77±0.27) (p=0.0043, Student's t-test). AUC_(0-30min)of the group to which compound 1 was intraperitoneally administered was14.60±0.46 and, as compared with the positive control group, thebronchoconstriction was significantly suppressed by 105% (p=0.0047,Student's t-test).

As shown in FIG. 2, AUC_(0-30min) of the bronchoconstriction in thepositive control group (19.61±0.75, mean value±standard error)significantly increased as compared with AUC_(0-30min) of the negativecontrol group (13.37±0.20) (p<0.0001, Student's t-test). AUC_(0-30min)of the group to which compound 1 was orally administered was 16.85±0.84and, as compared with the positive control group, thebronchoconstriction was significantly suppressed by 44% (p=0.0248,Student's t-test).

As shown in FIG. 3, AUC of airway hyperreactivity of the positivecontrol group (335.13±52.6, mean value±standard error) significantlyincreased as compared with AUC of the negative control group(184.7±27.5) (p=0.0182, Student's t-test). AUC of the group to whichcompound 1 was administered was 243.23±48.7 and, as compared with thepositive control group, airway hyperreactivity was suppressed by 60%.AUC of the group to which prednisolone was administered was 269.12±46.7and, as compared with the positive control group, airway hyperreactivitywas suppressed by 43%.

As shown in FIG. 4, eosinophil numbers in bronchoalveolar lavage fluidof the negative control group was 0.00±0.00×10⁵ per-mouse and, in thepositive control group, a significant increase of 2.77±0.46×10⁵ wasnoted (p=0.0009°, Aspin-Welch test). Eosinophil numbers in the group towhich compound 1 was administered and the group to which prednisolonewas administered per mouse were 0.92±0.26×10⁵ and 0.76±0.25×10⁵,respectively. As compared with the positive control group, eosinophilnumbers were significantly decreased by 67% in the group to whichcompound 1 was administered (p=0.0030, Student's t-test). In the groupto which prednisolone was administered, eosinophil numbers weresignificantly decreased by 73% (p=0.0015, Student's t-test).

From the above results, it has been suggested that a substance capableof suppressing the function involved in signal transduction of proteincomprising an amino acid sequence represented by SEQ ID NO: 11 is usefulas an agent for treatment of itching.

The medicament of the present invention is characterized in containing asubstance selected from a group consisting of the nitrogen-containingtricyclic compound represented by the formula (I) or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofand the hydrate and the solvate thereof as an active ingredient. Withregard to the medicament of the present invention, the above-mentionedsubstance which is an active ingredient may be administered as it isbut, usually, it is desirable to administer in a form of apharmaceutical composition containing the above-mentioned substancewhich is an active ingredient and one or more additives for thepreparation. Such a pharmaceutical composition is able to be prepared bya method which is known or common in the field of pharmaceuticalpreparation science. The medicament according to the present inventionin a form of a pharmaceutical composition may contain one or more otherpharmaceutically active ingredient(s). Incidentally, the pharmaceuticalof the present invention is able to be applied to mammals including ahuman being.

With regard to the administration route of the medicament of the presentinvention, there is no particular limitation and the most effectiveadministration route for the treatment and/or the prevention may beappropriately selected from oral administration and parenteraladministration such as intravenous injection. An example of thepharmaceutical preparation suitable for oral-administration is tabletsor the like and an example of the pharmaceutical preparation suitablefor parenteral administration is injection preparation or the like.

In the manufacture of a solid preparation such as tablet; it is possibleto use excipient such as lactose and mannitol; disintegrating agent suchas starch; lubricant such as magnesium stearate; binder such ashydroxypropyl cellulose; surfactant such as fatty acid ester;plasticizer such as glycerol or the like.

Among the preparation suitable for parenteral administration,preparation for administration into blood vessel such as injectionpreparation may be preferably prepared using an aqueous medium which isisotonic to human blood. For example, an injection preparation is ableto be prepared according to a conventional method as solution,suspension or dispersion together with an appropriate excipient using anaqueous medium selected from salt solution, glucose solution, a mixtureof salt and glucose solution, etc. For the manufacture of pharmaceuticalpreparation for parenteral use, it is possible to use one or moreadditive(s) for pharmaceutical preparations selected from diluent,flavor, antiseptic, excipient, disintegrating agent, lubricant, binder,surfactant, plasticizer, and the like.

There is no particular limitation for the dose and administeringfrequency of the pharmaceutical of the present invention but it ispossible to appropriately select depending upon various conditions suchas type of the above substance which is an active ingredient,administration route, object of treatment and/or prevention, age andbody weight of patient, nature of symptom and degree of severeness. Forexample, it is preferred to administer 0.1 to 100 mg/kg per day for anadult by dividing into three to four times a day. However, dose andadministering frequency as such may vary depending upon theabove-mentioned various conditions, etc.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is now more specifically illustrated by way of thefollowing Referential Examples and Examples, the scope of the presentinvention is not limited by these Examples, or the like.

Physicochemical data for each of the compounds in the followingReferential Examples were measured by the following instruments.

¹H-NMR: JOEL JNM-EX270 (270 MHz) or JEOL JNM-GX270 (270 MHz)

MS: Micromass LCT or Micromass Quatro (measured by an APCI method)

REFERENTIAL EXAMPLE 1 Synthesis of compound 1{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(4-methylpiperazin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine(30.0 g, 78.4 mmol) mentioned in the JP-A-7-61983 was dissolved in amixed solvent of chloroform (300 mL) and acetic acid (300 mL), then1-methylpiperazine (23.6 g, 236 mmol) and formaldehyde (37% aqueoussolution; 7.64 g, 94.1 mmol) were added thereto and the mixture washeated at 60° C. and stirred for 18 hours. After confirming the progressof the reaction by a thin-layer chromatography, a saturated aqueoussolution of sodium bicarbonate was added under cooling with ice andextracted with ethyl acetate. The organic layer was successively washedwith a saturated aqueous solution of sodium bicarbonate, water and asaturated aqueous solution of salt, dried over anhydrous sodium sulfateand concentrated in vacuo. The crystals separated therefrom weretriturated with ethyl acetate to give compound 1 (27.4 g, 55.4 mmol,yield: 71%).

APCI-MS: m/z 495 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 2.27 (s, 3H), 2.45 (m,8H), 2.60 (s, 3H), 2.63 (s, 3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H),3.38 (s, 2H), 5.34, (s, 2H), 6.00 (s., 1H), 6.57-6.66 (m, 2H), 6.79-7.00(m, 5H).

The corresponding fumarate was prepared according to the followingprocess.

Thus, the above compound 1 (15 g) was dissolved in methanol (110 mL) andfumaric acid (7.0 g, 2.0 equivalents) was added. A suspension wherefromcrystals were separated was once concentrated to dryness, acetonitrile(100 mL) was added and a suspension was stirred for not shorter than 1hour. After that, the crystals were filtered and dried in vacuo to givea difumarate of compound 1 (20.1 g, yield: 91%).

REFERENTIAL EXAMPLE 2 Synthesis of compound 2{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 1 was conductedusing 1,2,3,6-tetrahydropyridine instead of 1-methylpiperazine and,starting from2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinementioned in the JP-A-7-61983, compound 2 was obtained in the yield of20%.

APCI-MS: m/z 478 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.04 (m, 2H), 2.53 (t,J=5.7 Hz, 2H), 2.60 (s, 3H), 2.62 (s, 3H), 2.79 (q, J=7.5 Hz, 2H),2.86-3.02 (m, 6H), 3.45 (s, 2H), 5.33 (s, 2H), 5.64 (m, 1H), 5.74 (m,1H), 6.02 (s, 1H), 6.57-6.70 (m, 2H), 6.78-6.82 (m, 2H), 6.88 (s, 1H),6.95-7.00 (m, 2H).

REFERENTIAL EXAMPLE 3 Synthesis of Compound 3{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 1 was conductedusing pyrrolidine instead of 1-methylpiperazine and, starting from2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinementioned in the JP-A-7-61983, compound 3 was obtained in the yield of20%.

APCI-MS: m/z 466 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 1.78 (m, 4H), 2.50 (m,4H), 2.60 (s, 3H), 2.63 (s, 3H), 2.79 (q, J=7.5 Hz, 2H), 2.98 (m, 4H),3.50 (s, 2H), 5.34 (s, 2H), 6.02 (s, 1H), 6.58-6.66 (m, 2H), 6.79-6.81(m, 2H), 6.88 (s, 1H), 6.98-7.02 (m, 2H).

REFERENTIAL EXAMPLE 4 Synthesis of Compound 4{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-morpholinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 1 was conductedusing morpholine instead of 1-methylpiperazine and, starting from2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinementioned in the JP-A-7-61983, compound 4 was obtained in the yield of46%.

APCI-MS: m/z 482 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.43 (m, 4H), 2.60 (m,3H), 2.63 (m, 3H), 2.79 (q, J=7.5 Hz, 2H), 2.98 (m, 4H), 3.38 (s, 2H),3.69 (m, 4H), 5.34 (s, 2H), 6.07 (s, 1H), 6.58-6.67 (m, 2H), 6.78-6.81(m, 2H), 6.88 (s, 1H), 6.96-7.01 (m, 2H).

REFERENTIAL EXAMPLE 5 Synthesis of Compound 5 to Compound 12

2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine(19 mg, 0.050 mmol) mentioned in the JP-A-7-61983 was dissolved in amixed solvent of chloroform (0.30 mL) and acetic acid (0.30 mL), then achloroform solution of the corresponding R⁵R⁶NH (1.0 mol/L, 0.15 mL) andformaldehyde (37% aqueous solution, 0.005 mL) were added thereto and themixture was heated at 60° C. and stirred for 20 hours. After confirmingthe progress of the reaction by a thin-layer chromatography, the solventwas evaporated and the residue was dissolved in chloroform and washedwith water for two times. The organic layer was dried over anhydroussodium sulfate and concentrated. To the residue were added chloroform(0.50 mL) and N-methylisatoic anhydride polystyrene (manufactured byNova-Biochem, 0.15 mL) were added followed by stirring at roomtemperature for one night. The resin in the reaction mixture wasfiltered off and the residue was purified by means of ion-exchangechromatography (Bondecil SCX, manufactured by Varian, eluted with a 2mmol/L methanolic solution of ammonia) to give objective compound 5 tocompound 12.

Structures and analytical data (APCI-MS) of the compounds are shown inTable 1 and Table 14, respectively.

REFERENTIAL EXAMPLE 6 Synthesis of Compound 13{1-[8-(2-Ethyl-5,7-dimethyl-3H-imidazol[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-ylmethyl]-1-methyl-pyrrolidiniumiodide}

Compound 3 (11.4 g, 24.5 mmol) prepared in Referential Example 3 wasdissolved in dichloromethane (200 mL), methyl iodide (1.98 mL, 31.8mmol) was added thereto and the mixture was stirred at room temperaturefor 10 hours. After the reaction solution was concentrated in vacuo,ethyl acetate was added thereto. The resulting suspension was heated at60° C., stirred for 0.5 hour and then stirred at room temperature for 1hour. The solid separated out was filtered to give compound 13 (13.7 g,22.5 mmol, yield: 92%).

APCI-MS: m/z-480 ([M−I]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.6 Hz, 3H), 2.13 (br s, 2H), 2.25(br s, 2H), 2.58 (s, 3H), 2.62 (s, 2H), 2.79 (q, J=7.6 Hz, 2H), 2.85 (m,4H), 3.06 (s, 3H), 3.52 (br s, 2H), 3.83 (br s, 2H), 4.74 (s, 2H), 5.32(s, 2H), 6.76 (m, 2H), 6.88 (s, 1H), 6.95-7.18 (m, 4H), 7.43 (s, 1H).

REFERENTIAL EXAMPLE 7 Synthesis of Compound 14{2-(2,5-Dihydropyrrol-1-ylmethyl)-8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-di-benzo[b,f]azepine}

The operation similar to that in Referential Example 1 was conductedusing 2,5-dihydropyrrole instead of 1-methylpiperazine and, startingfrom2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinementioned in the JP-A-7-61983, compound 14 was obtained in the yield of82%.

APCI-MS: m/z 464 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.59 (s, 3H), 2.63 (s,3H), 2.79 (q, J=7.5 Hz, 2H), 2.97-3.1 (m, 4H), 3.45 (s, 4H), 3.70 (s,2H), 5.34 (s, 2H), 5.87 (s, 2H), 6.07 (s, 1H), 6.59 (d, J=8.7 Hz, 2H),6.63 (d, J=8.7 Hz, 2H), 6.75-6.85 (m, 2H), 6.88 (s, 1H), 7.00-7.05 (m,2H).

REFERENTIAL EXAMPLE 8 Synthesis of Compound 15<Methyl{N-8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-ylmethyl}-N-methylamino)acetate>

The operation similar to that in Referential Example 1 was conductedusing sarcosine methyl ester hydrochloride instead of 1-methylpiperazineand, starting from2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine,mentioned in the JP-A-7-61983, compound 15 was obtained in the yield of31%.

APCI-MS: m/z 498 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 2.36 (s, 3H), 2.60 (s,3H), 2.63 (s, 3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 3.23 (s, 2H),3.53 (s, 2H), 3.70 (s, 3H), 5.34 (s, 2H), 5.98 (s, 1H), 6.59-6.67 (m,2H), 6.82 (m, 2H), 6.88 (s, 1H), 6.97-7.02 (m, 2H).

REFERENTIAL EXAMPLE 9 Synthesis of Compound 16{Ethyl-1-[8-2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-ylmethyl]piperidine-4-carboxylate}

The operation similar to that in Referential Example 1 was conductedusing ethyl isonipecotate instead, of 1-methylpiperazine and, startingfrom2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinementioned in the JP-A-7-61983, compound 16 was obtained in the yield of60%.

APCI-MS: m/z 552 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.23 (t, J=7.0 Hz, 3H), 1.30 (t, J=7.6 Hz, 3H),1.68-1.90 (m, 6H), 1.97 (td, J=11.3, 2.7 Hz, 2H), 2.26 (m, 1H), 2.60 (s,3H), 2.62 (s, 3H), 2.79 (q, J=7.6 Hz, 2H), 2.83 (m, 2H), 2.98 (m, 4H),3.36 (s, 2H), 4.11 (q, J=7.0 Hz, 2H), 5.33 (s, 2H), 6.03 (s, 1H),6.57-6.66 (m, 2H), 6.78-6.82 (m, 2H), 6.88 (s, 1H), 6.94-6.99 (m, 2H).

REFERENTIAL EXAMPLE 10 Synthesis of Compound 17<2-{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-ylmethyl]-N-methylamino}ethanol>

Lithium aluminum hydride (15.7 mg, 0.38 mmol) was suspended intetrahydrofuran (0.3 mL) with stirring under cooling with ice. Compound15 (126 mg, 0.253 mmol) prepared in Referential Example 8 dissolved intetrahydrofuran (0.9 mL) was added thereto followed by stirring at roomtemperature for 1.5 hours. After confirming the progress of the reactionby means of thin-layer chromatography, water (0.016 mL), a 2 mol/Laqueous solution of sodium hydroxide (0.016 mL) and water (0.048 mL)were dropped thereinto successively with stirring. The precipitate wasfiltered off, the filtrate was concentrated and the resulting residuewas purified by an NH-silica gel chromatography (eluting solvent: ethylacetate) to give compound 17 (47.6 mg, 0.101 mmol, yield: 40%).

APCI-MS: m/z 470 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 1.7 (br s, 1H), 2.21 (s,3H), 2.57 (t, J=5.5 Hz, 2H), 2.60 (s, 3H), 2.63 (s, 3H), 2.80 (q, J=7.5Hz, 2H), 2.98 (m, 4H), 3.44 (s, 2H), 3.61 (t; J=5.5 Hz, 2H), 5.34 (s,2H), 5.99 (s, 1H), 6.59-6.6.7 (m, 2H), 6.81 (m, 2H), 6.88 (s, 1H),6.91-6.98 (m, 2H).

REFERENTIAL EXAMPLE 11 Synthesis of Compound 18<{1-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidin-4-yl}methanol>

Compound 16 was used instead of compound 15 and, in the manner similarto that in Referential Example 10, compound 18 was prepared in a yieldof 50%.

APCI-MS: m/z 510 [M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 1.24-1.74 (m, 6H), 1.91(m, 2H), 2.60 (s, 3H), 2.63 (s, 3H), 2.79 (q, J=7.6 Hz, 2H), 2.86-3.02(m, 6H), 3.37 (s, 2H), 3.48 (d, J=6.3 Hz, 2H), 5.34 (s, 2H), 5.98 (s,1H), 6.58-6.67 (m, 2H), 6.82 (m, 2H), 6.89 (s, 1H), 6.94-7.00 (m, 2H).

REFERENTIAL EXAMPLE 12 Synthesis of Compound 19<{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-+2-ylmethyl]-N-methylamino}aceticacid>

Compound 15 (151 mg, 0.303 mmol) prepared in Referential Example 8 wasdissolved in methanol (3.0 mL), a 1 mol/L methanol solution of sodiumhydroxide (1.5 mL) was added thereto and the mixture was heated at 60°C. and stirred for 9 hours. After confirming the progress of thereaction by a thin-layer chromatography, it was cooled to roomtemperature and pH was adjusted to 6.0 by addition of 4 mol/Lhydrochloric acid. The crystals separated out therefrom were filteredand dried in vacuo. The crystals were suspended in ethyl ether, stirredfor 1 hour under heating with refluxing and then stirred for 1 hour atroom temperature. The crystals were filtered and dried in vacuo to givecompound 19 (119 mg, 0.246 mmol, yield: 81%).

APCI-MS: m/z 483 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 2.34 (s, 3H),2.48-2.52 (s×2, 6H, overlapped with DMSO), 2.78 (q, J=7.4 Hz, 2H), 2.89(m, 4H), 3.11 (s, 2H), 3.66 (s, 2H), 5.29 (s, 2H), 6.75-7.02 (m, 7H),8.36 (s, 1H).

REFERENTIAL EXAMPLE 13 Synthesis of Compound 20{1-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidine-4-carboxylicacid}

The operation similar to that in Referential Example 12 was conductedusing compound 16 instead of compound 15 to give compound 20 in a yieldof 70%.

APCI-MS: m/z 524 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 1.52 (m, 2H), 1.75 (m,2H), 1.97 (m, 2H), 2.18 (m, 1H), 2.48-2.54 (s×2, 6H, overlapped withDMSO), 2.71-2.92 (m, 8H), 3.32 (s, 2H), 5.29 (s, 2H), 6.75-6.94 (m, 7H),8.23 (s, 1H).

REFERENTIAL EXAMPLE 14 Synthesis of Compound 21<{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-methylamino}acetonitrile>

Compound 13 (700 mg, 1.15 mmol) prepared in Referential Example 6 wasdissolved in chloroform (1.2 mL)., then methylaminoacetonitrile (368 mg,3.46 mmol) and triethylamine (0.561 mL, 4.03 mmol) were added theretoand the mixture was stirred for one night under refluxing. The reactionsolution was cooled to room temperature, a saturated aqueous solution ofsodium bicarbonate was added and the mixture was extracted withchloroform for three times. The organic layers were combined, washedwith a saturated aqueous saline solution, dried over anhydrous magnesiumsulfate and concentrated and the residue was purified by a silica gelchromatography (eluting solvent: methanol/chloroform=1/99). Ethanol wasadded to the concentrated residue of the fraction containing theobjective substance and the resulting suspension was stirred at 60° C.for 0.5 hour and then stirred at room temperature for 1 hour. Thecrystals separated out therefrom were filtered and dried in vacuo togive compound 21 (415 mg, 0.893 mmol, yield: 78%).

APCI-MS: m/z 465 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.42 (s, 3H), 2.60 (s,3H), 2.63 (s, 3H), 2.79 (q; J=7.5 Hz, 2H), 2.98 (m, 4H), 3.43 (s, 2H),3.48 (s, 2H), 5.34 (s, 2H), 6.10 (s, 1H), 6.58-6.69 (m, 2H), 6.78-6.83(m, 2H), 6.88 (s, 1H), 6.95-7.02 (m, 2H).

REFERENTIAL EXAMPLE 15 Synthesis of Compound 22{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-[2-(pyrrolidin-1-yl)ethyl]aminedifumarate}

Step 1

Compound 93 (1.25 g, 3.03 mmol) prepared in Referential Example 24 whichis mentioned later was dissolved in a mixed solvent of chloroform (54mL) and acetone (6 mL), then manganese dioxide (2.7 g, 31 mmol) wasadded thereto and the mixture was stirred for one night at roomtemperature. After confirming the progress of the reaction by athin-layer chromatography, the solid was filtered off using a Celite andthe filtrate was concentrated. Ethyl acetate was added to the residue,the resulting suspension was stirred for 0.5 hour under reflux, thencooled to room temperature and stirred for 0.5 hour. The crystalsseparated out therefrom were filtered and dried in vacuo to give8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboaldehyde(1.02 g, 2.48 mmol, yield: 82%).

APCI-MS: m/z 411 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.64 (s,3H), 2.80 (q, J=7.5 Hz, 2H), 2.99 (m, 2H), 3.06 (m, 2H), 5.37 (s; 2H),6.60-6.91 (m, 6H), 7.52-7.61 (m, 2H), 9.77 (s, 1H).

Step 2

8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboaldehyde(0.300 g, 0.73 mmol) prepared in step 1 was suspended in a mixed solventof tetrahydrofuran (10 mL) and chloroform (6 mL), then2-(pyrrolidin-1-yl)ethylamine (139 μL, 1.10 mmol) was added thereto andthe mixture was heated to reflux for 10 minutes. After that, thereaction solution was cooled to room temperature, sodiumtriacetoxyborohydride (464 mg, 2.19 mmol) was added thereto and themixture was stirred for 12 hours at room temperature. To the reactionsolution were added ethyl acetate and a 1 mol/L aqueous solution ofsodium hydroxide and the organic layer was dried over anhydrousmagnesium sulfate. After that, the solution was concentrated in vacuoand the residue was purified by a silica gel chromatography (elutingsolvent: chloroform/2 mol/L methanolic solution of ammonia=20/1) to giveN-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-[2-(pyrrolidin-1-yl)ethyl]amine(0.301 g, 0.592 mmol, yield: 81%). This was made into a fumarate by themethod similar to Referential Example 1 to give compound 22.

APCI-MS: m/z 509 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 1.65-1.85 (m, 4H),2.50 (s, 3H), 2.51 (s, 3H), 2.6-2.7 (m, 4H), 2.7-3.0 (m, 8H), 3.86 (s,2H), 5.29 (s, 2H), 6.55 (s, 4H), 6.75-6.95 (m, 6H), 7.0-7.15 (m, 2H),8.43 (s, 1H).

REFERENTIAL EXAMPLE 16 Synthesis of Compound 23{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-(2-methoxyethyl)aminemonofumarate}

The operation similar to that in step 2 of Referential Example 15 wasconducted using 2-methoxyethylamine instead of2-(pyrrolidin-1-yl)ethylamine to giveN-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-(2-methoxyethyl)aminein a yield of 78%. This was converted to a fumarate by the mannersimilar to that in Referential Example 1 to give compound 23.

APCI-MS: m/z 470 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 2.50 (s, 3H), 2.51 (s,3H), 2.80 (q, J=7.4 Hz, 2H), 2.8-3.0 (m, 6H), 3.24 (s, 3H), 3.49 (t,J=6.5 Hz, 2H), 3.80 (s, 2H), 5.29 (s, 2H), 6.48 (s, 2H), 6.84 (d, J=8.1Hz, 1H), 6.85-7.0 (m, 4H), 7.0-7.1 (m, 2H), 8.43 (s, 1H).

REFERENTIAL EXAMPLE 17 Synthesis of Compound 24<2-{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]aminoethanol0.5 fumarate}

The operation similar to that in step 2 of Referential Example 15 wasconducted using 2-ethanolamine instead of 2-(pyrrolidin-1-yl)ethylamineto give2-{[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]amino}ethanolin a yield of 39%.

This was converted to a fumarate by the manner similar to that inReferential Example 1 to give compound 24.

APCI-MS: m/z 456 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 2.50 (s, 3H), 2.51 (s,3H), 2.70-2.75 (m, 2H), 2.77 (q, J=7.4 Hz, 2H), 2.85-2.9 (m, 4H), 3.55(t, J=5.5 Hz, 2H), 3.78 (s, 2H), 5.29 (s, 2H), 6.44 (s, 1H), 6.79 (dd,J=1.5 Hz, 8.3 Hz, 1H), 6.85-6.95 (m, 4H), 7.0-7.1 (m, 2H), 8.39 (s, 1H).

REFERENTIAL EXAMPLE 18 Synthesis of compound 25<{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]methyl}aminemonofumarate>

Compound 13 (0.300 g, 0.516 mmol) prepared in Referential Example 6 wasdissolved in a 7 mol/L methanolic solution of ammonia (5 mL), sealed andheated at 80° C. for 48 hours. After that, the reaction solution wasconcentrated in vacuo. The residue was purified by a silica gelchromatography (eluting solvent: chloroform/2 mol/L methanolic ammoniasolution=20/1) to give{[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]-azepin-2-yl]methyl}amine(0.135 g, 0.219 mol, yield: 64%).

This was converted to a fumarate by the manner similar to that inReferential Example 1 to give compound 25.

APCI-MS: m/z 412 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 2.50 (s, 3H), 2.51 (s,3H), 2.77 (q, J=7.4 Hz, 2H), 2.85-2.9 (m, 4H), 3.81 (s, 2H), 5.29 (s,2H), 6.42 (s, 2H), 6.8-7.0 (m, 5H), 7.0-7.15 (m, 2H), 8.46 (s, 1H).

REFERENTIAL EXAMPLE 19 Synthesis of Compound 26{N-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-N-methyl-N-(2H-tetrazol-5-ylmethyl)amine}

Compound 13 (667 mg, 1.10 mmol) prepared in Referential Example 6 wasdissolved in chloroform (11 mL), thenN-methyl-N-(2-trityl-2H-tetrazol-5-ylmethyl)amine (390 mg, 1.10 mmol)prepared in Referential Example 22 and triethylamine (0.31 mL, 2.3 mmol)were added thereto and the mixture was stirred at 60° C. for one night.The reaction solution was cooled to room temperature, a saturatedaqueous solution of sodium bicarbonate was added and the mixture wasextracted with chloroform for three times. The organic layers werecombined, washed with a saturated saline solution, dried over anhydrousmagnesium sulfate and concentrated. The residue was passed throughsilica gel (eluting solvent: methanol/chloroform=2/98) to removestarting point components and concentrated. To the residue were addedacetone (1.9 mL), water (1.9 mL) and acetic acid (1.9 mL) and themixture was stirred at 60° C. for 1.5 hours. The reaction solution wascooled down to 0° C., separated substances were filtered off, thefiltrate was concentrated and the resulting residue was recrystallizedfrom ethanol to give compound 26 (66.7 mg, 0.131 mmol, yield: 12%).

APCI-MS: m/z 508 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.32 (t, J=5.0 Hz, 3H), 2.58 (s, 3H), 2.63 (s,3H); 2.75-2.79 (m, 7H), 2.81 (q, J=5.0 Hz, 2H), 4.08 (s, 2H), 4.28 (s,2H), 5.34 (s, 2H), 6.37 (s, 1H), 6.46 (d, J=8.1 Hz, 1H), 6.58 (d, J=8.1Hz, 1H), 6.72-6.80 (m, 2H), 6.84-6.94 (m, 3H).

REFERENTIAL EXAMPLE 20 Synthesis of Compound 27{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-[4-(2H-tetrazol-5-yl)piperidin-1-ylmethyl]-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 14 was conductedusing piperidine-4-carbonitrile instead of methylaminoacetonitrile togive1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-piperidine-4-carbonitrile.

The resulting

1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-piperidine-4-carbonitrile(0.252 g, 0.500 mmol) was dissolved in toluene (4 mL), thentrimethylsilyl azide (0.13 mL, 1.00 mmol) and dibutyltin oxide (12.4 mg,0.05 mmol) were added and the mixture was heated with stirring at 110°C. for 22 hours. The reaction solution was concentrated in vacuo andethanol was added to the residue. The resulting suspension was heated toreflux for 0.5 hour and the solid was filtered to give compound 27(0.110 g, 0.200 mmol, yield: 40%).

APCI-MS: m/z 548 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.22 (t, J=7.4 Hz, 3H), 1.65-1.85 (m, 2H),1.9-2.05 (m, 2H), 2.2-2.35 (m, 2H), 2.48 (s, 3H), 2.58 (s, 3H), 2.77 (q,J=7.4 Hz, 2H), 2.85-3.05 (m, 7H), 3.52 (s, 2H), 5.29 (s, 2H), 6.85-7.05(m, 8H), 8.36 (s, 1H).

REFERENTIAL EXAMPLE 21 Synthesis of Compounds 28 to 90

Step 1

1-(10,11-Dihydro-5H-dibenz[b,f]azepin-2-ylmethyl)-1-methylpiperidiniumiodide (0.015 g, 0.050 mmol) was dissolved in dimethyl formamide (0.50mL), then a chloroform solution of the corresponding YH (wherein Y hasthe same meaning as defined above) (1.0 mmol/L, 0.060 mL) and lithiumhydroxide monohydrate (0.070 g) were added and the mixture was stirredat room temperature for 20 hours. After confirming the progress of thereaction by a thin-layer chromatography, the solvent is evaporated, theresidue was dissolved in dichloromethane and the resulting solution waswashed with water for three times. The organic layer was dried overanhydrous sodium sulfate and concentrated, chloroform (0.60 mL) andN-methylisatoic anhydride polystyrene (manufactured by Nova Biochem,0.15 mL) were added thereto and the mixture was stirred at roomtemperature for one night. The resin in the reaction-mixture wasfiltered off, the filtrate was concentrated and the residue was purifiedby an ion-exchange chromatography (Bondecil SCX, manufactured by Barian,eluted with 2 mol/L methanolic solution of ammonia) to give variousintermediates corresponding to compound (IV) in the Producing Process 1.

Step 2

The operation similar to that in Referential Example 5 was conducted togive desired compounds 28 to 90 from various intermediates correspondingto compound (IV) in the Producing Process 1 obtained in step 1 and thecorresponding R⁵R⁶NH (wherein R⁵ and R⁶ each have the same meaning asdefined above, respectively). Incidentally, compounds 41, 42, 48 and 89were isolated as oxalates.

Structures and analytical data (APCI-MS) of compounds 28 to 87 are shownin Tables 2 to 6. Analytical data (¹H-NMR) of compounds 29, 30, 36, 41,42, 48, 53, 54, 60, 65, 66, 72, 77, 78 and 84 are shown below.

Compound 29:

{2-(Benzimidazol-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.0-2.1 (m, 2H), 2.44 (t, J=5.6 Hz, 2H),2.75-2.85 (m, 2H), 2.9-3.0 (m, 4H), 3.32 (s, 2H), 5.31 (s, 2H), 5.5-5.8(m, 2H), 6.8-7.1 (m, 6H), 7.1-7.3 (m, 2H), 7.56 (d, J=77.1 Hz, 1H), 7.62(d, J=7.4 Hz, 1H), 8.28 (s, 1H), 8.35 (s, 1H).

Compound 30:

{2-(Benzimidazol-1-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.5-1.7 (m, 4H), 2.3-2.5 (m, 4H), 2.8-3.0 (m,4H), 3.39 (s, 2H), 5.31 (s, 2H), 6.8-6.95 (m, 4H), 6.95-7.0 (m, 2H),7.1-7.3 (m, 2H), 7.55 (d, J=8.9 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 8.26(s, 1H), 8.34 (s, 1H).

Compound 36:

{2-(Benzimidazo-1-ylmethyl)-8-morpholinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.2-2.4 (m, 4H), 2.8-3.0 (m, 4H), 3.27 (s,2H), 3.5-3.6 (m, 4H), 5.30 (s, 2H), 6.7-7.1 (m, 6H), 7.1-7.25 (m, 2H),7.54 (d, J=7.6 Hz, 1H), 7.62 (d, J=7.6 Hz, 1H), 8.28 (s, 1H), 8.34 (s,1H).

Compound 41:

{2-(2-Phenylbenzimidazol-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinemonooxalate}

¹H NMR (DMSO-d₆) δ (ppm): 2.2-2.5 (m, 2H), 2.7-3.0 (m, 4H), 3.0-3.2 (m,2H), 3.4-3.6 (m, 2H), 4.05 (s, 2H), 5.45 (s, 2H), 5.69 (m, 1H), 5.85 (m,1H), 6.6-6.8 (m, 2H), 6.88 (d, J=8.3 Hz, 1H), 6.97 (d, J=7.9 Hz, 1H),7.05-7.2 (m, 2H), 7.2-7.5 (m, 2H), 7.5-7.7 (m, 4H), 7.7-7.85 (m, 3H),8.54 (s, 1H).

Compound 42:

{2-(2-Phenylbenzimidazol-1-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinemonooxalate}

¹H NMR (DMSO-d₆) δ (ppm): 1.8-2.0 (m, 4H), 2.8-3.0 (m, 4H), 3.0-3.2 (m,4H), 4.12 (s, 2H), 5.45 (s, 2H), 6.6-6.7 (m, 2H), 6.88 (d, J=8.1 Hz,1H), 6.96 (d, J=7.8 Hz, 1H), 7.1-7.2 (m, 2H), 7.2-7.3 (m, 2H), 7.4-7.6(m, 4H), 7.6-7.8 (m, 3H), 8.53 (s, 1H).

Compound 48:

{2-Morpholinomethyl-8-(2-phenylbenzimidazol-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinemonooxalate}

¹H NMR (DMSO-d₆) δ (ppm): 2.7-3.0 (m, 8H), 3.6-3.8 (m, 4H), 3.83 (s,2H), 5.42 (s, 2H), 6.65-6.7 (m, 2H), 6.85 (d, J=8.2 Hz, 1H), 6.92 (d,J=8.1 Hz, 1H), 7.0-7.1 (m, 2H), 7.2-7.3 (m, 2H), 7.4-7.6 (m, 4H),7.65-7.8 (m, 3H), 8.44 (s, 1H).

Compound 53:

{2-(2-Methylbenzimidazol-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.0-2.1 (m, 2H), 2.45 (t, J=5.6 Hz, 2H), 2.54(s, 3H), 2.75-2.85 (m, 2H), 2.85-3.0 (m, 4H), 3.35 (s, 2H), 5.28 (s,2H), 5.55-5.75 (m, 2H), 6.8-7.0 (m, 6H), 7.1-7.2 (m; 2H), 7.4-7.6 (m,2H), 8.28 (s, 1H).

Compound 54:

{2-(2-Methylbenzimidazol-1-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.5-1.8 (m, 4H), 2.3-2.5 (m, 4H), 2.54 (s,3H), 2.8-3.0 (m, 4H), 3.39 (s, 2H), 5.28 (s, 2H), 6.7-6.9 (m, 6H),7.1-7.2 (m, 2H), 7.3-7.5 (m, 2H), 8.25 (s, 1H).

Compound 60:

{2-(2-Methylbenzimidazo-1-ylmethyl)-8-morpholinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.2-2.4 (m, 4H), 2.49 (s, 3H), 2.8-3.0 (m,4H), 3.28 (s, 2H), 3.5-3.6 (m, 4H), 5.28 (s, 2H), 6.8-7.0 (m, 6H),7.1-7.2 (m, 2H), 7.5-7.6 (m, 2H), 8.28 (s, 1H).

Compound 65:

{2-(5,6-Dimethylbenzimidazol-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.0-2.1 (m, 2H), 2.2-2.4 (m, 6H), 2.45 (t,J=5.2 Hz, 2H), 2.75-2.85 (m, 2H), 2.85-3.05 (m, 4H), 3.30 (s, 2H), 5.24(s, 2H), 5.6-5.7 (m, 2H), 6.8-7.0 (m, 6H), 7.31 (s, 1H), 7.40 (s, 1H),8.17 (s, 1H), 8.27 (s, 1H).

Compound 66:

{2-(5,6-Dimethylbenzimidazol-1-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.5-1.8 (m, 4H), 2.27 (s, 3H), 2.28 (s, 3H),2.3-2.4 (m, 4H), 2.8-3.0 (m, 4H), 3.39 (s, 2H), 5.24 (s, 2H), 6.8-7.0(m, 6H), 7.30 (s, 1H), 7.40 (s, 1H), 8.16 (s, 1H), 8.24 (s, 1H).

Compound 72:

{2-(5,6-Dimethylbenzimidazol-1-ylmethyl)-8-morpholinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 2.2-2.4 (m, 10H), 2.8-3.0 (m, 4H), 3.28 (s,2H), 3.5-3.6 (m, 4H), 5.24 (s, 2H), 6.8-7.0 (m, 6H), 7.30 (s, 1H), 7.39(s, 1H), 8.16 (s, 1H), 8.28 (s, 1H).

Compound 77:

{2-(2-Ethylbenzimidazol-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.28 (t, J=7.4 Hz, 3H), 1.95-2.05 (m, 2H),2.43 (t, J=5.4 Hz, 2H), 2.6-3.0 (m, 8H), 3.32 (s, 2H), 5.28 (s, 2H),5.1-5.5 (m, 2H), 6.75-7.0 (m, 6H), 7.1-7.25 (m, 2H), 7.47 (m, 1H), 7.55(m, 1H), 8.26 (s, 1H).

Compound 78:

{2-(2-Ethylbenzimidazol-1-ylmethyl)-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.28 (t, J=7.4 Hz, 3H), 1.6-1.8 (m, 4H),2.3-2.4 (m, 4H), 2.8-3.0 (m, 6H), 3.32 (s, 2H), 5.28 (s, 2H), 6.7-7.0(m, 6H), 7.0-7.2 (m, 2H), 7.46 (m, 1H), 7.54 (m, 1H), 8.23 (s, 1H).

Compound 84:

{2-(2-Ethylbenzimidazo-1-ylmethyl)-8-morpholinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

¹H NMR (DMSO-d₆) δ (ppm): 1.28 (t, J=7.4 Hz, 3H), 2.2-2.4 (m, 4H),2.8-3.0 (m, 6H), 3.27 (s, 2H), 3.5-3.6 (m, 4H), 5.27 (s, 2H), 6.7-7.0(m, 6H), 7.1-7.2 (m, 2H), 7.47 (m, 1H), 7.55 (m, 1H), 8.26 (s, 1H).

Structures of compounds 88 to 90 are shown in Table 7 and analyticaldata thereof (APCI-MS and ¹H-NMR) are shown below.

Compound 88:

{2-(Imidazo[4,5-b]pyridin-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

APCI-MS: m/z 422 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 2.0-2.1 (m, 2H), 2.44 (t, J=5.4 Hz, 2H),2.75-2.8 (m, 2H), 2.8-3.0 (m, 4H), 3.30 (s, 2H), 5.33 (s, 2H), 5.5-5.6(m, 2H), 6.8-7.0 (m, 4H), 7.0-7.05 (m, 2H), 7.27 (dd, J=4.7 Hz, 8.0 Hz,1H), 8.06 (d, J=8.0 Hz, 1H), 8.27 (s, 1H), 8.37 (d, J=4.7 Hz, 1H), 8.54(s, 1H).

Compound 89:

{2-(Imidazo[4,5-b]pyridin-3-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepinemonooxalate}

APCI-MS: m/z 422 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 2.2-2.3 (m, 2H), 2.9-3.0 (m, 4H), 3.4-3.5 (m,2H), 3.60 (t, J=6.8 Hz, 2H), 4.05 (s, 2H), 5.37 (s, 2H), 5.67 (d, J=10.8Hz, 1H), 5.85 (d, J=10.8 Hz, 1H), 6.9-7.0 (m, 2H), 7.0-7.1 (m, 4H), 7.25(dd, J=5.4, 8.1 Hz, 1H), 8.01 (d, J=8.1 Hz, 1H), 8.40 (d, J=5.4 Hz, 1H),8.55 (s, 1H), 8.62 (s, 1H).

Compound 90:

{2-(Imidazo[4,5-c]pyridin-1-ylmethyl)-8-(1,2,5,6-tetrahydropyridin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

APCI-MS: m/z 422 ([M+H]⁺)

¹H-NMR (CDCl₃) δ (ppm): 2.1-2.2 (m, 2H), 2.56 (t, J=5.7 Hz, 2H), 2.8-2.9(m, 2H), 3.0-3.1 (m, 4H), 3.48 (s, 2H), 5.30 (s, 2H), 5.67 (d, J=10.5Hz, 1H), 5.73 (d, J=10.5 Hz, 1H), 6.08 (s, 1H), 6.65-6.75 (m, 2H),6.95-7.0 (m, 2H), 7.0-7.05 (m, 2H), 7.71 (d, J=5.4 Hz, 1H), 8.02 (s,1H), 8.45 (d, J=5.4 Hz, 1H), 8.78 (s, 1H).

REFERENTIAL EXAMPLE 22 Synthesis ofN-methyl-N-(2-trityl-2H-tetrazol-5-ylmethyl)amine

2-Trityl-2H-tetrazol-5-ylmethanol (2.00 g, 5.84 mmol),N-methyl-2-nitrobenzenesulfonamide (1.64 g, 7.59 mmol) andtriphenylphosphine (1.53 g, 5.84 mmol) were dissolved in a mixed solventof tetrahydrofuran (30 mL) and toluene (20 mL), then a solution ofdiethyl azodicarboxylate in toluene (40%, 2.65 mL, 5.84 mmol) was addedthereto and the mixture was stirred at room temperature for throughout anight. After the mixture was passed through a silica gel column (elutingsolvent: ethyl acetate/hexane=40/60) to remove original-pointcomponents, acetone (5 mL) and acetonitrile (25 mL) were added to theresidue prepared by concentrating in vacuo.

Mercaptoacetic acid (0.73 mL, 11 mmol) and1,8-diazabicyclo[5,4,0]undec-7-ene (3.1 mL, 21 mmol) were added to theresulting suspension and the mixture was stirred at 60° C. for 7 hours.The reaction solution was concentrated and the residue was dissolved inethyl acetate. The resulting solution was washed with a saturatedaqueous solution of sodium bicarbonate and a saturated saline solutionsuccessively, dried over anhydrous magnesium sulfate and concentrated.The residue was purified by a silica gel chromatography (elutingsolvent: triethylamine/ethyl acetate=1/99) to prepareN-methyl-N-(2-trityl-2H-tetrazol-5-yl)methylamine (396 mg, 1.11 mmol,yield: 19.0%).

¹H NMR (CDCl₃) δ (ppm): 2.45 (s, 3H), 4.07 (s, 2H), 7.07-7.36 (m, 15H).

REFERENTIAL EXAMPLE 23 Synthesis of Compound 92{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]acetate}

Compound 13 (7.98 g, 13.1 mmol) prepared in Referential Example 6 wasdissolved in dimethyl sulfoxide (87 mL), lithium acetate (4.33 g, 65.7mL) was added thereto and the mixture was stirred at 70° C. for 2 days.The reaction solution was diluted with ethyl acetate, washed with water(for three times) and a saturated saline solution successively, driedover anhydrous magnesium sulfate and concentrated. The residue waspurified using a silica gel chromatography (eluting solvent: ethylacetate) to concentrate a fraction containing the objective product,ethanol was added to the residue and the resulting suspension wasstirred at room temperature for 0.5 hour. The crystals separated outtherefrom were filtered to give compound 92 (2.87 g, 6.31 mmol, yield:48%).

APCI-MS: m/z 455 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.06 (s, 3H), 2.60 (s,3H), 2.62 (s, 3H), 2.79 (q, J=7.5 Hz, 2H), 2.98 (m, 4H), 4.98 (s, 2H),5.34 (s, 2H), 6.13 (s, 1H), 6.58-6.83 (m, 4H), 6.88 (s, 1H), 7.01-7.07(m, 2H).

REFERENTIAL EXAMPLE 24 Synthesis of compound 93{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]methanol}

Compound 92 (2.79 g, 6.14 mmol) prepared in Referential Example 23 wassuspended in tetrahydrofuran (61 mL), then a methanolic solution ofsodium methoxide (28%, 6.2 mL, 31 mmol) was added and the mixture wasstirred at room temperature for 3.5 hours. After confirming the progressof the reaction by a thin-layer chromatography, water was added to thereaction solution and the mixture was stirred at room temperature for0.5 hour. The crystals precipitated therefrom were filtered, dried invacuo and suspended in ethanol and the suspension was stirred for 1 hourunder reflux and then stirred for another 1 hour at room temperature.The crystals, separated out therefrom were filtered and dried in vacuoto give compound 93 (2.04 g, 4.95 mmol, yield: 81%).

APCI-MS: m/z 413 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 1.56 (t, J+=5.6 Hz, 1H),2.60 (s., 3H), 2.63 (s, 3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 4.55(d, J=5.6 Hz, 2H), 5.34 (s, 2H), 6.03 (s, 1H), 6.59-6.85 (m, 3H), 6.88(s, 1H), 7.03 (m, 2H).

REFERENTIAL EXAMPLE 25 Synthesis of Compound 94{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-methoxymethyl-10,11-dihydro-5H-dibenz[b,f]azepine}

Methanol (20 μL, 0.50 mmol) was added to a suspension of sodium hydride(55%, 11 mg, 0.25 mmol) in tetrahydrofuran (0.40 mL) and the mixture wasstirred at room temperature for 20 minutes. After that, the reactionsolution was added to a suspension of compound 13 (30 mg, 0.050 mmol)prepared in Referential Example 6 in tetrahydrofuran (0.20 mL) and thereaction was conducted at 60° C. for 3.5 hours. After concentrating thereaction solution, the residue was dissolved in chloroform, theresulting solution was washed with water and a saturated saline solutionsuccessively, dried over anhydrous magnesium sulfate and concentrated.The residue was purified by a silica gel chromatography (elutingsolvent: ethyl acetate/hexane/triethylamine=45/50/5) to give compound 94(6.5 mg, 15 mmol, yield: 30%).

APCI-MS: m/z 427 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 3.36 (s, 3H), 4.32 (s, 2H),5.34 (s, 2H), 6.09 (s, 1H), 6.58-6.82 (m, 4H), 6.88 (s, 1H), 7.01 (m,2H).

REFERENTIAL EXAMPLE 26 Synthesis of Compound 95{2-Allyloxymethyl-8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation smilar to that in Referential Example 25 was conductedusing allyl alcohol instead of methanol to give compound 95 in a yieldof 34%.

APCI-MS: m/z 453 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 4.00 (dt, J=5.6, 1.5 Hz, 2H),4.39 (s, 2H), 5.19 (dq, J=10.2, 1.5 Hz, 1H), 5.29 (dq, J=17.0, 1.5 Hz,1H), 5.34 (s, 2H), 5.95 (m, 1H), 6.10 (s, 1H), 6.58-6.83 (m, 4H), 6.88(s, 1H), 7.03 (m, 2H).

REFERENTIAL EXAMPLE 27 Synthesis of Compound 96{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2-methoxyethoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing 2-methoxyethanol-instead of methanol to give compound 96 in ayield of 9.3%.

APCI-MS: m/z 495 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q J=7.5 Hz, 2H), 2.98 (m, 4H), 3.38 (s, 3H), 3.57 (m, 4H),4.44 (s, 2H), 5.34 (s, 2H), 6.01 (s, 1H), 6.62 (d, J=8.6 Hz, 1H), 6.67(d, J=8.1 Hz, 1H), 6.82 (m, 2H), 6.88 (s, 1H), 7.00-7.06 (m, 2H).

REFERENTIAL EXAMPLE 28 Synthesis of Compound 97{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2,2,2-trifluoroethoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing 2,2,2-trifluoroethanol instead of methanol to give compound 97 ina yield of 64%.

APCI-MS: m/z 495 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.6 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 3.78 (q, J=8.7 Hz, 2H), 4.54(s, 2H), 5.34 (s, 2H), 6.24 (s, 1H), 6.60 (d, J=7.8 Hz, 1H), 6.71 (d,J=8.1 Hz, 1H), 6.76-6.82 (m, 2H), 6.89 (s, 1H), 6.98-7.04 (m, 2H)

REFERENTIAL EXAMPLE 29 Synthesis of Compound 98{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2-methylpropoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing 2-methyl-1-propanol instead of methanol to give compound 98 in ayield of 11%.

APCI-MS: m/z 469 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 0.91 (d, J=6.7 Hz, 6H), 1.30 (t, J=7.4 Hz, 3H),1.89 (m, 1H), 2.60 (s, 3H), 2.63 (s, 3H), 2.79 (q, J=7.4 Hz, 2H), 2.99(m, 4H), 3.20 (d, J=6.5 Hz, 2H), 4.37 (s, 2H), 5.34 (s, 2H), 6.01 (s,1H), 6.60 (d, J=8.9 Hz, 1H), 6.67 (d, J=7.8 Hz, 1H), 6.81 (m, 2H), 6.88(s, 1H), 6.98-7.05 (m, 2H).

REFERENTIAL EXAMPLE 30 Synthesis of Compound 99{2-Benzyloxymethyl-8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing benzyl alcohol instead of methanol to give compound 99 in a yieldof 78%.

APCI-MS: m/z 503 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J 7.5 Hz, 3H), 2.60 (s, 3H), 2.62 (s,2H), 2.79 (q, J=7.5 Hz, 2H), 2.97 (m, 4H), 4.42 (s, 2H), 4.53 (s, 2H),5.33 (s, 2H), 6.20 (s, 1H), 6.59 (d, J=7.9 Hz, 1H), 6.69 (d, J=7.9 Hz,1H), 6.78 (m, 2H), 6.88 (s, 1H), 7.02 (m, 2H), 7.26-7.36 (m, 5H).

REFERENTIAL EXAMPLE 31 Synthesis of Compound 100{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2-phenylethoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing 2-phenylethanol instead of methanol to give compound 100 in ayield of 38%.

APCI-MS: m/z-517 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q, J=7.5 Hz, 2H), 2.91 (t, J=7.2 Hz, 2H), 2.97 (m, 4H), 3.66(t, J=7.2 Hz, 2H), 4.39 (s, 2H), 5.34 (s, 2H), 6.08 (s, 1H), 6.60 (d,J=8.7 Hz, 1H), 6.66 (d, J=8.1 Hz, 1H), 6.80 (m, 2H), 6.88 (s, 1H),6.94-7.01 (m, 2H), 7.19-7.30 (m, 5H).

REFERENTIAL EXAMPLE 32 Synthesis of Compound 101{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(pyridin-2-ylmethoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing pyridin-2-ylmethanol instead of methanol to give compound 101 in ayield of 65%.

APCI-MS: m/z 504 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q, J=7.5 Hz, 2H), 2.98 (m, 4H), 4.52 (s, 2H), 4.66 (s, 2H),5.34 (s, 2H), 6.25 (s, 1H), 6.60 (d, J=7.9 Hz, 1H), 6.70 (d, J=7.9 Hz,1H), 6.76-6.81 (m, 2H), 6.88 (s, 1H), 7.03-7.08 (m, 2H), 7.18 (br dd,J=7.6, 4.8 Hz, 1H), 7.47 (d, J=7.9 Hz, 1H), 7.68 (td, J=7.7, 1.8 Hz,1H), 8.54 (br d, J=4.8 Hz, 1H).

REFERENTIAL EXAMPLE 33 Synthesis of Compound 102{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(furan-2-ylmethoxymethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

The operation similar to that in Referential Example 25 was conductedusing furan-2-ylmethanol instead of methanol to give compound 102 in ayield of 77%.

APCI-MS: m/z 493 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.62 (s,3H), 2.79 (q, J=7.5 Hz, 2H), 2.97 (m, 4H), 4.41 (s, 2H), 4.45 (s, 2H),5.33 (s, 2H), 6.21 (br s, 1H), 6.31 (dd, J=3.1, 0.8 Hz, 1H), 6.33 (dd,J=3.1, 1.8 Hz, 1H), 6.58 (d, J=8.1 Hz, 1H), 6.69 (d, J=7.9 Hz, 1H),6.75-6.80 (m, 2H), 6.88 (s; 1H), 7.00-7.04 (m, 2H), 7.40 (dd, J=1.8, 0.8Hz, 1H).

REFERENTIAL EXAMPLE 34 Synthesis of Compound 103{8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-carbonitrile}

8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboaldehyde(650 mg, 1.58 mmol) prepared in step 1 of Referential Example 15 wassuspended in acetonitrile (16 mL), then hydroxylamine hydrochloride (153mg, 2.38 mmol), triethylamine (0.331 mL, 2.38 mmol) and phthalicanhydride (328 mg, 2.21 mmol) were added thereto and the mixture wasstirred at 80° C. for one night. The reaction solution was concentrated,the residue was dissolved in chloroform, the resulting solution waswashed with an aqueous ammonia solution (3%) and a saturated salinesolution successively, dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by a silica gel chromatography(eluting solvent: methanol/chloroform=1/99), the fraction containing theobjective product was concentrated, ethanol was added to the residue andthe resulting suspension was stirred at 60° C. for 0.5 hour and stirredat room temperature for 1 hour. The crystals precipitated therefrom werefiltered and dried in vacuo to give compound 103 (440 mg, 1.08 mmol,yield: 68%).

APCI-MS: m/z 408 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t+J=7.6 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.79 (q, J=7.6 Hz, 2H), 2.98 (m, 4H), 5.36 (s, 2H), 6.48 (s, 1H),6.63-6.90 (m, 5H), 7.28-7.33 (m, 2H).

REFERENTIAL EXAMPLE 35 Synthesis of Compound 104{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2H-tetrazol-5-yl)-10,11-dihydro-5H-dibenz[b,f]azepine}

Compound 103 prepared in Referential Example 34 was used and, by thesimilar manner as in the latter part of Referential Example 20, compound104 was obtained in a yield of 72%

APCI-MS: m/z 451 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.24 (t, J=7.4 Hz, 3H), 2.48-2.53 (s×2, 6H,overlapped with DMSO), 2.80 (q, J=7.4 Hz, 2H), 2.86-3.02 (m, 4H), 5.32(s, 2H), 6.83 (dd, J=8.1, 2.1 Hz, 1H), 6.91-6.98 (m, 3H), 7.10 (d, J=9.0Hz, 1H), 7.65-7.70 (m, 2H), 8.20 (s, 1H).

REFERENTIAL EXAMPLE 36 Synthesis of Compound 105{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]acetonitrile}

Compound 13 (2.04 g, 3.36 mmol) prepared in Referential Example 6 wasdissolved in dimethylformamide (17 mL), sodium cyanide (361 mg, 7.37mmol) was added thereto and the mixture was stirred at 50° C. for 10hours. The reaction solution was cooled to room temperature, dilutedwith ethyl acetate, washed with a 2 mol/L aqueous solution of sodiumhydroxide, water (two times) and a saturated saline solutionsuccessively, dried over anhydrous magnesium sulfate and concentrated.The residue was recrystallized from ethanol to give compound 105 (751mg, 1.78 mmol, yield: 53%).

APCI-MS: m/z 422 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J 7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.79 (q, J=7.5 Hz, 2H), 2.99 (m, 4H), 3.62 (s, 2H), 5.34 (s, 2H),6.01 (s, 1H), 6.59-6.71 (m, 2H), 6.80-6.84 (m, 2H), 6.88 (s, 1H),6.95-7.01 (+m, 2H).

REFERENTIAL EXAMPLE 37 Synthesis of Compound 106{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-8-(2H-tetrazol-5-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

Compound 105 prepared in Referential Example 36 was used and, by thesimilar manner as in the latter part of Referential Example 20, compound106 was obtained in a yield of 76%

APCI-MS: m/z 465 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.22 (t, J=7.6 Hz, 3H), 2.48-2.52 (s×2, 6H,overlapped with DMSO), 2.78 (q, J=7.6 Hz, 2H), 2.86 (m, 4H), 4.11 (s,2H), 5.28 (s, 2H), 6.75-6.94 (m, 7H), 8.32 (br s, 1H).

REFERENTIAL EXAMPLE 38 Synthesis of Compound 107{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]aceticacid}

Compound 105 (247 mg, 0.586 mmol) prepared in Referential Example 36 wassuspended in ethanol (12 mL), sodium hydroxide (938 mg, 23.5 nmol) wasadded thereto and the mixture was stirred for 3 hours under a conditionof heating to reflux. After confirming the progress of the reaction by athin-layer chromatography, the reaction solution was cooled to roomtemperature and pH was adjusted to 5 with 1 mol/L hydrochloric acid. Thecrystals separated out therefrom were filtered, dried in vacuo,suspended in ethanol, stirred at 60° C. for 0.5 hour and stirred at roomtemperature for 1 hour. The crystals separated out were filtered anddried in vacuo to give compound 107 (122 mg, 0.243 mmol, yield: 41%).

APCI-MS: m/z 441 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.5 Hz, 3H), 2.48-2.53 (s×2, 6H,overlapped with DMSO), 2.78 (q, J=7.5 Hz, 2H), 2.87 (br s, 4H), 3.38 (s,2H), 5.38 (s, 2H), 6.74-6.94 (m, 7H), 8.27 (s, 1H), 12.15 (br s, 1H).

REFERENTIAL EXAMPLE 39 Synthesis of Compound 108 {Methyl[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl-sulfanyl]acetate}

Compound 13 (1.04 g, 1.71 mmol) prepared in Referential Example 6 wasdissolved in chloroform (17 mL), then methyl mercaptoacetate (0.199 mL,2.23 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.384 mL, 2.57 mmol)were added thereto and the mixture was stirred at 40° C. for 7 hours.The reaction solution was concentrated, the residue was purified by asilica gel chromatography (eluting solvent: methanol/chloroform=1/99)and a fraction containing compound was concentrated. Ethanol was addedto the residue and the resulting suspension was stirred at 60° C. for0.5 hour and then at room temperature for 1 hour. The crystals separatedout were filtered to give compound 108 (628 mg, 1.25 mmol, yield: 73%).

APCI-MS: m/z 501 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.30 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q, J=7.5 Hz, 2H), 2.98 (m, 4H), 3.09 (s, 2H), 3.72 (s, 3H),3.73 (s, 2H), 5.34 (s, 2H), 6.01 (s, 1H), 6.59-6.67 (m, 2H), 6.82 (m,2H), 6.88 (s, 1H), 6.96-7.03 (m, 2H)

REFERENTIAL EXAMPLE 40 Synthesis of Compound 109{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethylsulfanyl]aceticacid}

Compound 108 (350 mg, 0.699 mmol) prepared in Referential Example 39 wasused and, by the similar manner as in Referential Example 12, compound109 was obtained in a yield of 38%

APCI-MS: m/z 487 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm) 1.16 (t, J=7.4 Hz, 3H), 2.42-2.50 (s×2, 6H,overlapped with DMSO), 2.81 (q, J=7.4 Hz, 2H), 2.88 (m, 6H), 3.49 (s,2H), 5.22 (s, 2H), 6.67-6.89 (m, 7H), 8.18 (s, 1H).

REFERENTIAL EXAMPLE 41 Synthesis of Compound 110 {Ethyl8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylate}

Step 1:

1-(10,11-Dihydro-5H-dibenz[b,f]azepin-2-ylmethyl)-1-methylpiperidiniumiodide (6.68 g, 15.4 mmol) was dissolved in dimethyl sulfoxide (110 mL),lithium acetate (5.07 g, 76.9 mmol) was added thereto and the mixturewas stirred at 70° C. for 2 days. The reaction solution was diluted withethyl acetate, washed with water (for three times) and a saturatedsaline solution successively, dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by a silica gel chromatography(eluting solvent: ethyl acetate/hexane=30/70) to give(10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl) acetate (2.85 g, 10.7mmol, yielde: 69%).

APCI-MS: m/z 268 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 2.07 (s, 3H), 3.07 (br s, 4H), 4.99 (s, 2H),6.05 (br s, 1H), 6.66-6.85 (m, 3H), 7.02-7.11 (m, 4H).

Step 2:

(10,11-Dihydro-5H-dibenz[b,f]azepin-2-ylmethyl) acetate (2.85 g, 10.7mmol) prepared in the step 1 was suspended in methanol (110 mL), then amethanolic solution of sodium methoxide (38%, 1.14 mL, 5.36 mmol) wasadded thereto and the mixture was stirred at room temperature for 1hour. The reaction solution was concentrated, a saturated salinesolution and chloroform were added to the residue and extraction wasconducted with chloroform for three times. The organic layers werecombined, dried over anhydrous magnesium sulfate and concentrated. Theresidue was recrystallized from diisopropyl ether to give10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethanol (1.73 g, 7.68 mmol,yield: 72%).

APCI-MS: m/z 226 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.49 (t, J=5.8 Hz, 1H), 3.08 (br s, 4H), 4.57(d, J=5.8 Hz, 2H), 6.02 (br s, 1H), 6.66-6.87 (m, 3H), 7.02-7.11 (m,4H).

Step 3:

10,11-Dihydro-5H-dibenz[b,f]azepin-2-ylmethanol (6.1 g, 70 mmol)prepared in the step 2 was dissolved in chloroform (77 mL), thenmanganese dioxide (4.55 g, 46.1 mmol) was added thereto and the mixturewas stirred at room temperature for 8 hours. The reaction solution wasfiltered through Celite and the filtrate was concentrated. The residuewas purifed by a silica gel chromatography (eluting solvent: ethylacetate/hexane=20/80) togive-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboaldehyde (1.15 g, 5.15mmol, yield: 67%).

APCI-MS: m/z 224 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 3.11 (m, 4H), 6.49 (br s, 1H), 6.87-6.91 (m,3H), 7.07-7.17 (m, 2H), 7.55-7.62 (m, 2H), 9.88 (s, 1H).

Step 4:

10,11-Dihydro-5H-dibenz[b,f]azepin-2-carboaldehyde (665 mg, 2.98 mmol)prepared in the step 3 was dissolved in a mixed solvent of acetonitrile(18 mL) and water (18 mL), then dimethyl sulfoxide (2.1 mL, 30 mmol),sodium dihydrogen phosphate (1.43 g, 11.9 mmol) and sodium chlorite (404mg, 4.47 mmol) were added thereto and the mixture was stirred at 50° C.for 4 hours. Ethyl acetate and water were added to the reaction solutionand extraction was conducted with ethyl acetate for two times. Theorganic layers were combined, washed with water, dried over anhydrousmagnesium sulfate and concentrated. A mixed solvent (3:1) of ethylacetate and hexane was added to the residue and the resulting suspensionwas stirred at 60° C. for 0.5 hour and stirred at room temperature for 1hour. The crystals separated out were filtered to give10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylic acid (598 mg, 2.50 mmol,yield: 84%).

APCI-MS: m/z 240 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 3.11 (m, 4H), 6.39 (br s, 1H), 6.77-6.80 (m,3H), 7.06-7.16 (m, 2H), 7.79-7.84 (m, 2H).

Step 5:

10,11-Dihydro-5H-dibenz[b,f]azepin-2-carboxylic, acid (426 mg, 1.78mmol) prepared in the step 4 was dissolved in ethanol (8.9 mL), thionylchloride (0.26 mL, 3.6 mmol) was added thereto and the mixture wasstirred for 5 hours with heating to reflux. The reaction solution wasconcentrated, chloroform and a saturated aqueous solution of sodiumbicarbonate were added thereto and extraction with chloroform wasconducted for three times. The organic layers were combined, washed witha saturated saline solution, dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by a silica gel chromatography(eluting solvent: ethyl acetate/hexane=10/90) to give ethyl10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylate (383 mg, 1.43 mmol,yield: 81-W).

APCI-MS: m/z 268 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.37 (t; J=7.0 Hz, 3H), 3.09 (m, 4H), 4.33 (q,J=7.0 Hz, 2H), 6.34 (br s, 1H), 6.69-6.86 (m, 3H), 7.04-7.14 (m, 2H),6.72-6.78 (m, 2H).

Step 6:

Ethyl 10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylate (443 mg, 1.66mmol) prepared in the step 5 was dissolved in a mixed solvent ofchloroform (8.3 mL) and acetic acid (8.3 mL), then piperidine (0.573 mL,5.80 mmol) and paraformaldehyde (149 mg, 4.97 mmol) were added theretoand the mixture was heated at 60° C. and stirred for 1.5 days. Thereaction solution was concentrated, ethyl acetate and a saturatedaqueous saline solution were added thereto and extraction with ethylacetate was conducted. The organic layers were combined, washed with asaturated saline solution, dried over anhydrous sodium sulfate andconcentrated. The residue was purified by a silica gel chromatography(eluting solvent: ethyl acetate/hexane/triethylamine=70/25/5) and afraction containing the objective substance was concentrated. Theresidue was subjected to trituration with diethyl ether to give ethyl8-piperidinomethyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylate (249mg, 0.683 mmol, yield: 41%).

APCI-MS: m/z 365 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.34-1.46 (m, 5H), 1.67 (m, 4H), 2.36 (br s,4H), 3.08 (m, 4H), 3.38 (s, 2H), 4.33 (q, J=7.1 Hz, 2H), 6.32 (s, 1H);6.67-6.74 (m, 2H), 6.99-7.06 (m, 2H), 7.72-7.76 (m, 2H).

Step 7

Ethyl8-piperidinomethyl-10,11-dihydro-5H-dibenz[b,f]azepine-2-carboxylate(231 mg, 0.634 mmol) prepared in the step 6 was dissolved indichloromethane (3.2 mL), methyl iodide (59.2 μL, 0.951 mmol) was addedthereto and the mixture was stirred at room temperature for one night.The reaction solution was concentrated in vacuo to give1-(8-ethoxycarbonyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl)-1-methylpiperidiniumiodide (321 mg, 0.634 mmol, yield: 100%).

¹H NMR (CDCl₃) δ (ppm): 1.37 (t, J=7.1 Hz, 3H), 1.75-1.95 (m, 6H), 2.96(br s, 4H), 3.11 (s, 3H), 3.50 (m, 2H), 3.70 (m, 2H), 4.32 (q, J=7.1 Hz,2H), 4.90 (s, 2H), 7.14-7.35 (m, 4H), 7.49 (s, 1H), 7.71 (m, 2H).

Step 8:

2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridine (180 mg, 1.03 mmol) wasdissolved in dimethylformamide (0.60 mL), sodium hydride (55%, 33.6 mg,0.770 mmol) was added thereto with stirring by dividing into severaltimes and the mixture was stirred at 50° C. for 0.5 hour. The reactionsolution was cooled down to room temperature, then a solution of1-(8-ethoxycarbonyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl)-1-methylpiperidiniumiodide (1.30 mg, 0.256 mmol) prepared in the step 7 dissolved indimethylformamide (1.2 mL) was added thereto and the mixture was stirredat room temperature for 1 hour. The reaction solution was diluted withethyl acetate, washed with water, water and a saturated aqueous salinesolution successively, dried over anhydrous magnesium sulfate andconcentrated. The residue was purified by a silica gel chromatography(eluting solvent: methanol/chloroform=1/99) and a fraction containingthe objective substance was concentrated. Diethyl ether was added to theresidue and the mixture was stirred for 0.5 hours with heating to refluxand, after that, stirred at room temperature for 1 hour. The crystalsseparated out therefrom were filtered to give compound 110 (76.7 mg,0.169 mmol, yield: 66%).

APCI-MS: m/z 455 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.6 Hz, 3H), 1.36 (t, J=7.1 Hz, 3H),2.60 (s, 3H), 2.63 (s, 3H), 2.80 (q, J=7.6 Hz, 2H), 2.97 (m, 2H), 3.40(m, 2H), 4.32 (q, J=7.1 Hz, 2H), 5.36 (s, 2H), 6.35 (s, 1H), 6.64-6.71(m, 2H), 6.82-6.90 (m, 3H), 7.70-7.74 (m, 2H).

REFERENTIAL EXAMPLE 42 Synthesis of compound 111{8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-carboxylicacid}

The operation similar to that inn Referential Example 12 was conductedusing compound 110 (900 mg, 1.98 mmol) prepared in Referential Example41 to give compound 111 in a yield of 97%.

APCI-MS: m/z 427 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.24 (t, J=7.5 Hz, 3H), 2.51-2.54 (s x 2, 6H,overlapped with DMSO), 2.82-2.99 (m, 6H), 5.37 (s, 2H), 6.84-7.03 (m,5H), 7.58 (m, 2H), 8.87 (br s, 1H), 12.25 (br s, 1H).

REFERENTIAL EXAMPLE 43 Synthesis of Compound 112{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl](4-methylpiperazin-1-yl)methanone}

Compound 111 (100 mg, 0.234 mmol) prepared in Referential Example 42 wasdissolved in a mixed solvent of dimethylformamide (2.3 mL) andtetrahydrofuran (4.6 mL), then 4-methylpiperazine (39 μL, 0.352 mmol),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide monohydrochloride (89.7mg, 0.468 mmol) and 1-hydroxybenzotriazole (35.8 mg, 0.234 mmol) wereadded thereto and the mixture was stirred at room temperature for 8hours. After confirming the progress of the reaction by a thin-layerchromatography, the reaction solution was concentrated. The residue wasdissolved in chloroform and the resulting solution was washed with water(for two times), a saturated sodium bicarbonate solution and a saturatedsaline solution successively, dried over anhydrous magnesium sulfate andconcentrated. Diethyl ether was added to the residue, the resultingsuspension was stirred at room temperature for 1 hour and solid wasfiltered to give compound 112 (47.7 mg, 0.0938 mmol, yield: 40%).

APCI-MS: m/z 509 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 2.33 (s, 3H), 2.43 (brs, 4H), 2.60 (s, 3H), 2.63 (s, 3H), 2.80 (q, J=7.5 Hz, 2H), 2.99 (m,4H), 3.66 (br s, 4H), 5.35 (s, 2H), 6.18 (s, 1H), 6.62-6.69 (m, 2H),6.83 (m, 2H), 6.85 (s, 1-H), 7.10-7.15 (m, 2H).

REFERENTIAL EXAMPLE 44 Synthesis of Compound 113{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl](pyrrolidine-1-yl)methanone}

The operation similar to that in Referential Example 43 was conductedusing pyrrolidine instead of 4-methylpiperazine to give compound 113 ina yield of 90%.

APCI-MS: m/z 480 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 1.88 (br s, 4H), 2.60(s, 3H), 2.63 (s, 3H), 2.80 (q, J=7.5 Hz, 2H), 2.99 (m, 4H), 3.56 (m,4H), 5.35 (s, 2H), 6.19 (s, 1H), 6.62-6.69° (m, 2H), 6.81-6.86 (m, 2H),6.89 (s, 1H), 7.24-7.29 (m, 2H).

REFERENTIAL EXAMPLE 45 Synthesis of compound 114{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl](4-hydroxypiperidino)methanone}

The operation similar to that in Referential Example 43 was conductedusing 4-piperidinol instead of 4-methylpiperazine to give compound 114in a yield of 62%.

APCI-MS: m/z 510 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.0 Hz, 3H), 1.48-1.58 (m, 2H),1.86-1.97 (m, 2H), 2.60 (s, 3H), 2.63 (s, 2H), 2.80 (q, J=7.0 Hz, 2H),2.99 (m, 4H), 3.22-3.33 (m, 2H), −3.91-4.00 (m, 3H), 5.36 (s, 2H), 6.21(s, 1H), 6.62-6.70 (m, 2H), 6.81-6.85 (m, 2H), 6.89 (s, 1H), 7.08-7.14(m, 2H).

REFERENTIAL EXAMPLE 46 Synthesis of Compound 115{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-carboxylicacid (2-hydroxyethyl)amide}

The operation similar to that in Referential Example 43 was conductedusing ethanolamine instead of 4-methylpiperazine to give compound 115 ina yield of 82%.

APCI-MS: m/z 470 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 1.71 (br s, 1H), 2.60(s, 3H), 2.63 (s, 3H), 2.79 (q, J=7.5 Hz, 2H), 2.97 (m, 4H), 3.59 (m,2H), 3.81 (t, J=9.6 Hz, 2H), 5.35 (s, 2H), 6.41 (s, 1H), 6.54 (t, J=5.6Hz, 1H), 6.63-6.71 (m, 2H), 6.80-6.84 (m, 2H), 6.99 (s, 1H), 7.44-7.48(m, 2H).

REFERENTIAL EXAMPLE 47 Synthesis of compound 116{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-carboxylicacid[2-(pyrolidin-1-yl)ethyl]amide}

The operation similar to that in Referential Example 43′ was conductedusing 2-(pyrrolidine-1-yl)ethylamine instead of 4-methylpiperazine togive compound 116 in a yield of 92%.

APCI-MS: m/z 523 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.6 Hz, 3H), 1.78 (m, 4H), 1.57 (m,4H), 2.60 (s, 3H), 2.63 (s, 3H), 2.70 (t, J=5.9 Hz, 2H), 2.79 (q, J=7.6Hz, 2H), 2.97 (m, 2H), 3.04 (m, 2H), 3.53 (q, J=5.7 Hz, 2H), 5.35 (s,2H), 6.30 (s, 1H), 6.63-6.72 (m, 3H), 6.83 (m, 2H), 6.89 (s, 1H),7.45-7.52 (m, 2H).

REFERENTIAL EXAMPLE 48 Synthesis of Compound 117{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl](morpholino)methanone)

The operation similar to that in Referential Example 43 was conductedusing morpholine instead of 4-methylpiperazine to give compound 117 in ayield of 98%.

APCI-MS: m/z 496 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q J=7.5 Hz, 2H), 2.99 (m, 4H), 3.66 (m, 8H), 5.35 (s, 2H),6.22 (s, 1H), 6.62-6.71 (m, 2H), 6.81-6.86 (m, 2H), 6.89 (s, 1H),7.10-7.16 (m, 2H).

REFERENTIAL EXAMPLE 49 Synthesis of Compound 118{8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-carboxylicacid bis(2-hydroxyethyl)amide}

The operation similar to that in Referential Example 43 was conductedusing 2-(2-hydroxyethylamino)ethanol instead of 4-methylpiperazine togive compound 118 in a yield of 38%.

APCI-MS: m/z 514 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.21 (t, J=7.5 Hz, 3H), 2.60 (s, 3H), 2.63 (s,3H), 2.80 (q, J=7.5 Hz, 2H), 2.98 (m, 4H), 3.23 (br s, 2H), 3.63 (br s,4H), 3.87 (br s, 4H), 5.35 (s, 2H), 6.20 (s, 1H), 6.62-6.69 (m, 2H),6.83 (m, 2H), 6.89 (s, 1H), 7.24-7.29 (m, 2H).

REFERENTIAL EXAMPLE 50 Synthesis of Compound 119{8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine-2-carboxylicacid amide}

The operation similar to that in Referential Example 43 was conductedusing ammonia instead of 4-methylpiperazine to give compound 119 in ayield of 57%.

APCI-MS: m/z 426 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 2.48-2.52 (s×2, 6H,overlapped with DMSO), 2.78 (q, J=7.4 Hz, 2H), 2.92 (br q, J=7.3 Hz,4H), 5.31 (s, 2H), 6.78-7.00 (m, 6H), 7.52-7.65 (m, 3H), 8.68 (s, 1H).

REFERENTIAL EXAMPLE 51 Synthesis of Compound 120{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-8-(pyrrolidin-1-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepine}

Compound 3 prepared in Referential Example 3 (400 mg, 0.876 mmol) wasdissolved in acetic acid (8.8 mL), then paraformaldehyde (0.47 g, 16mmol) and sodium cyanoborohydride (2.2 g, 10 mmol) were added theretoand the mixture was stirred at room temperature for 5 hours. Chloroformand a saturated sodium bicarbonate solution were added to the reactionsolution and the aqueous layer was extracted with chloroform twice. Theorganic layer was washed with a saturated saline solution, dried overanhydrous magnesium sulfate and concentrated. The residue was purifiedby an NH-silica gel chromatography (eluting solvent:chloroform/hexane=50/50) to give compound 120 (342 mg, 0.713 mmol,yield: 81%).

APCI-MS: m/z 480 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.31 (t, J=7.5 Hz, 3H), 1.76 (m, 4H), 2.47 (m,4H), 2.58 (s, 3H), 2.62 (s, 3H), 2.78 (q, J=7.5 Hz, 2H), 3.06 (m, 4H),3.29 (s, 3H), 3.50 (s, 2H), 5.35 (s, 2H), 6.83-6.98 (m, 5H), 7.02-7.08(m, 2H).

REFERENTIAL EXAMPLE 52 Synthesis of Compound 121{1-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidine-4-carboxylic acid}

Step 1:

Compound 16 (1.20, 2.18 mmol) prepared in Referential Example 9 wasdissolved in acetic acid (16 mL), then paraformaldehyde (0.73 g, 21.8mmol) and sodium cyanoborbhydride (0.58 g, 8.70 mmol) were added theretoand the mixture was stirred at room temperature for 15 hours. Ethylacetate and a 1 mol/L aqueous solution of sodium hydroxide were added tothe reaction solution and the aqueous layer was extracted with ethylacetate. The organic layer was dried over anhydrous magnesium sulfateand the solvent was evaporated in vacuo. The residue was purified by anNH-silica gel chromatography (eluting solvent: a mixed solvent ofhexane-ethyl acetate) to give ethyl1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidine-4-carboxylate(1.25 g, 2.18 mmol, yield: 100%).

APCI-MS: m/z 566 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 1.31 (t, J=7.6 Hz, 3H),1.6-2.0 (m, 6H), 2.23 (m, 1H), 2.58 (s, 3H), 2.62 (s, 3H), 2.73 (q,J=7.4 Hz, 2H) 2.75-2.9 (m, 2H), 3.0-3.15 (m, 4H), 3.28 (s, 3H), 3.36 (s,2H), 4.10 (q, J=7.6 Hz, 2H), 5.34 (s, 2H), 6.8-7.1 (m, 7H).

Step 2:

The operation similar to that in Referential Example 12 was conductedusing ethyl1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidine-4-carboxylateprepared in the step 1 to give compound 121 in a yield of 42%.

APCI-MS: m/z 538 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.23 (t, J=7.4 Hz, 3H), 1.5-1.8 (m, 2H),1.8-2.0 (m, 2H), 2.2-2.4 (m, 2H), 2.49 (s, 3H), 2.50 (s, 3H), 2.78 (q,J=7.4 Hz, 2H), 2.8-3.05 (m, 8H), 3.22 (s, 2H), 3.5-3.9 (m, 2H), 5.34 (s,2H), 6.85 (dd, J=2.0, 8.4 Hz, 1H), 6.93 (s, 1H), 6.94 (d, J=2.0 Hz, 1H),7.0-7.2 (m, 4H).

REFERENTIAL EXAMPLE 53 Synthesis of compound 122{2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-8-[4-(2H-tetrazol-5-yl)piperidinomethyl]-10,11-dihydro-5H-dibenz[b,f]azepine monohydrochloride}

The operation similar to that in the step 1 of Referential Example 52was conducted using1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]-piperidine-4-carbonitrileprepared in Referential Example 20 to prepare1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]-azepin-2-ylmethyl]piperidine-4-carbonitrilein a yield of 92%. The operation similar to that in the latter part ofReferential Example 20 was conducted using the above compound to prepare2-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5′-methyl-8-[4-(2H-tetrazol-5-yl)piperidinomethyl]-10,11-dihydro-5H-dibenz[b,f]azepinein a yield of 10%. This was dissolved in chloroform, a solution of 4mol/L of hydrogen chloride in ethyl acetate was added thereto and thesolid separated out therefrom were filtered to prepare compound 122.

APCI-MS: m/z 562 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.28 (t, J=7.6 Hz, 3H), 2.0-2.5 (m, 4H), 2.58(s, 3H), 2.63 (s, 3H), 2.9-3.2 (m, 8H), 3.2-3.3 (m, 4H), 3.4-3.6 (m,2H), 4.17 (s, 2H), 5.56 (s, 2H), 7.0-7.2 (m, 4H), 7.2-7.4 (m, 3H), 10.79(s, 1H).

REFERENTIAL EXAMPLE 54 Synthesis of Compound 123{1-[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidin-4-ylmethanol}

Ethyl1-[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-ylmethyl]piperidine-4-carboxylate(0.61 g, 1.08 mmol) prepared in the step 1 of Referential Example 52 wasdissolved in dichloromethane (10 mL), cooled at −78° C. and stirred. A 1mol/L solution of diisopropylaluminum hydride in toluene (3.20 mL, 3.20mmol) was added to the reaction solution at the same temperature and themixture was stirred for 3 hours at the same temperature and, after that,at room temperature for 10 minutes. A saturated aqueous solution ofRochelle salt: and ethyl acetate were added to the reaction solution andthe mixture was stirred for 30 minutes. The aqueous layer was extractedwith ethyl acetate, the organic layer was dried over anhydrous magnesiumsulfate and the solvent was evaporated in vacuo. The residue wassubjected to recrystallization from ethyl acetate to prepare compound123 (0.26 g, 0.50 mmol, yield: 46%).

APCI-MS: m/z 524 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.0-1.15 (m, 2H), 1.23 (t, J=7.5 Hz, 3H),1.25-1.3 (m, 1H), 1.5-1.65 (m, 2H), 1.7-1.9 (m, 2H), 2.49 (s, 3H), 2.50(s, 3H), 2.75 (q, J=7.5 Hz, 2H), 2.95-3.05 (m, 4H), 3.15-3.25 (m, 5H),3.25-3.50 (m, 4H), 5.32 (s, 2H), 6.81 (dd, J=2.0, 8.5 Hz, 1H), 6.90-7.05(m, 6H).

REFERENTIAL EXAMPLE 55 Synthesis of Compound 124[2-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-8-(2H-tetrazol-5-yl)-10,11-dihydro-5H-dibenz[b,f]azepine]

The operation similar to that in the step 1 of Referential Example 52was conducted using compound 103 prepared in Referential Example 34 toprepare8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepine-2-carbonitrilein a yield of 83%.

The operation similar to that in the latter part of Referential Example20 was conducted using the above compound to prepare compound 124 in ayield of 20%.

APCI-MS: m/z 465 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.24 (t, J=7.7 Hz, 3H), 2.50 (s, 3H), 2.51 (s,3H), 2.80 (q, J=7.7 Hz, 2H), 3.0-3.1 (m, 2H), 3.3-3.35 (m, 2H), 3.40 (s,3H), 5.38 (s, 2H), 6.90 (dd, J=2.2, 8.4 Hz, 1H), 6.95 (s, 1H), 7.02 (d,J=2.2 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 7.24 (d, J=8.4 Hz, 2H), 7.75 (d,J=2.2 Hz, 1H), 7.79 (dd, J=2.2, 8.4 Hz, 1H).

REFERENTIAL EXAMPLE 56 Synthesis of Compound 125{[8-(2-Ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]aceticacid}

The operation similar to that in step 1 of Referential Example 52 wasconducted using compound 105 prepared in Referential Example 36 toprepare[8-(2-ethyl-5,7-dimethyl-3H-imidazo[4,5-b]pyridin-3-ylmethyl)-5-methyl-10,11-dihydro-5H-dibenz[b,f]azepin-2-yl]acetonitrilein a yield of 94%.

The operation similar to that in Referential Example 38 was conductedusing the above compound to give compound 125 in a yield of 86%.

APCI-MS: m/z 455 ([M+H]⁺)

¹H NMR (DMSO-d₆) δ (ppm): 1.22 (t, J=7.3 Hz, 3H), 2.49 (s, 3H), 2.50 (s,3H), 2.75 (q, J=7.3 Hz, 2H), 2.9-3.1 (m, 4H), 3.19 (s, 3H), 3.42 (5,2H), 5.32 (s, 2H), 6.81 (d, J=8.1 Hz, 1H)}, 6.9-7.05 (m, 6H).

REFERENTIAL EXAMPLE 57 Synthesis of Compound 91{1,4-Bis[4-(3-chlorobenzylamino)-6-cyclopropylcarbonyl-7,8-dihydro-5H-pyrido[4,3-d]pyrimidin-2-yl]piperazine}

Compound 91 was synthesized according to the following step 1 to step 8.

A commercially-available compound (A) (100 g, 0.335 mol) was dissolvedin ethanol (1,500-mL), then urea (100 g, 1.67 mol) and sodium methoxide(227 g, 1.18 mol) were added thereto and the mixture was made to reactfor 24 hours under the condition of heating to reflux. Progress of thereaction was confirmed by a thin-layer chromatography and, after cooled,crystals separated out therefrom were filtered. The crystals weresuspended in water, hydrochloric acid (6 mol/L) was added thereto and pHwas adjusted to 6.0. Stirring was further conducted at room temperaturefor 1 hour and the crystals separated out therefrom were filtered anddried in vacuo to prepare compound (B) (60 g, yield: 70%).

Step 2:

Phosphorus oxychloride (300 mL) was added to compound (B) (30.0 g, 0.116mol) prepared in the step 1 and the mixture was stirred under a heatingcondition for 5 hours. After confirming the progress of the reaction bya thin-layer chromatography, an excessive phosphorus oxychloride wasevaporated in vacuo. After that, 2-propanol (300 mL) was added to theresidue and a suspension containing the crystals separated out therefromwas stirred under the condition of heating to reflux for 1 hour andfurther stirred at room temperature for 1 hour. The crystals separatedout were filtered and dried in vacuo to prepare compound (C) (33 g,yield: 85%).

Step 3:

Compound (C) (35.0 g, 0.106 mol) prepared in the step 2 was dissolved in1,2-dichloroethane (850 mL), then triethylamine (14.9 mL, 0.107 mol) and1-chloroethyl chloroformate (34.1 mL, 0.316 mol) were added thereto andthe mixture was stirred under a condition of heating to reflux for 5hours. After confirming the progress of the reaction by a thin-layerchromatography, the reaction mixture was cooled, washed with water and asaturated aqueous saline solution successively and dried over anhydrousmagnesium sulfate. The resulting solution was concentrated and theresidue was purified by a column chromatography (silica gel,n-hexane:ethyl acetate=3:1). The product was dissolved in methanol (850mL) and stirred under a condition of heating to reflux for 1 hour. Afterconfirming the progress of the reaction by a thin-layer chromatography,it was concentrated to dryness to prepare compound (D) (23.5 g, yield:95%).

Step 4:

Compound (D) (11.8 g, 49.1 mmol) prepared in Step 3 was dissolved indichloromethane (300 mL), then cyclopropanecarbonyl chloride (5.4 mL,1.2 equivalents) and triethylamine (20.4 mL, 3.0 equivalents) were addedthereto and the mixture was stirred at room temperature for 1 hour. Theresulting reaction solution was washed with water and a saturated sodiumbicarbonate solution and dried over magnesium sulfate. After evaporatingthe solvent, diisopropyl ether was added to the residue and thesuspension was stirred for not shorter than 1 hour. After that, thecrystals separated out therefrom were filtered and dried in vacuo toprepare compound (E) (12.5 g, yield: 94%).

Step 5:

Compound (E) (12.5 g, 45.9 mmol) prepared in the step 4 was dissolved intetrahydrofuran (400 mL), then triethylamine (19.2 ml, 3 equivalents)and 3-chlorobenzylamine (11.2 mL, 2 equivalents) were added and themixture was stirred at 40° C. for 20 hours. The salt separated outtherefrom was removed by filtration and the solvent was evaporated. Theresidue was purified by a chromatography(chloroform:methanol=100:1→40:1) and a mixed solvent of hexane/ethylacetate (3:1) was added to a concentrated residue of a fractioncontaining the objective substance so that the crystals were separatedout. The suspension containing the crystals was stirred for 1 hour andthe crystals separated out therefrom were filtered and dried in vacuo toprepare compound (F) (11.9 g, yield: 69%).

Step 6:

Compound (F) (5.0 g, 13.3 mmol) prepared in the step-5 was dissolved indioxane (100 mL), then tert-butyl 1-piperazinecarboxylate (4.9 g, 2equivalents) and sodium carbonate (14.0 g, 10 equivalents) were addedthereto and the mixture was, stirred at 90° C. for 3 days. The resultingreaction solution was filtered to remove-sodium carbonate, extractionwas conducted by addition of water and chloroform to the filtrate andthe organic layer was dried over magnesium sulfate. After evaporation ofthe solvent, a mixed solvent of hexane/ethyl acetate (3:1) was added andthe suspension was stirred for 1 hour. After that, the crystalsseparated out therefrom were filtered and dried in vacuo to preparecompound (G) (6.4 g, yield: 92%).

Step 7

A 20% solution of trifluoroacetic acid in dichloromethane (50 mL) wasadded to compound (G) (6.3 g, 12.0 mmol) prepared in the step 6 and themixture was stirred at room temperature for 1 hour. After evaporatingthe solvent from the reaction solution, diisopropyl ether was added tothe residue and the resulting suspension was stirred for 1 hour. Afterthat, the crystals separated out therefrom were filtered and dried invacuo to prepare compound (H) (4.9 g, yield: 97%)

Step 8

Compound (H) (3.8 g, 8.90 mmol) prepared in the step 7 and compound (F)(4.5 g, 1.05 equivalent) prepared in the step 5 were dissolved indioxane (100 mL), sodium carbonate (10.6 g, 10 equivalents) was addedand the mixture was stirred at 90° C. for 1 week. The resulting reactionsolution was filtered to remove sodium carbonate, water was added to thefiltrate and extraction was conducted with chloroform. The organic layerwas dried over magnesium sulfate, the solvent was evaporated and theresidue was purified by a column chromatography (ethylacetate:triethylamine=10:1). A mixed solvent of hexane/ethyl acetate(3:1) was added to a concentrated residue of a fraction containing theobejective substance and the resulting suspension was stirred for 1hour. After that, the crystals separated out therefrom were filtered anddried in vacuo to prepare compound 91 (1.0 g, yield: 23%).

APCI-MS: m/z 767 ([M+H]⁺)

¹H NMR (CDCl₃) δ (ppm): 0.7-0.9 (m, 4H), 1.0-1.1 (m, 4H), 1.7-1.9 (m,2H), 2.6-2.8 (m, 4H), 3.75 (s, 8H), 3.8-4.0 (m, 4H), 4.3-4.4 (m, 4H),4.6-4.7 (m, 4H), 4.8-4.9 (m, 2H), 7.1-7.3 (m, 8H).

REFERENTIAL EXAMPLE 58 Construction of a Host-Vector System

(1) Construction of Gal4-ER Expression Plasmid pGERbsrR2

pSV2bsr (manufactured by Kaken Seiyaku) was cleaved with PvuII and EcoRIand subjected to a Klenow treatment to prepare a PvuII (blunt end)-EcoRI(blunt end) fragment of 2.6 kb.

ERαAF2 in pM containing the Gal4-ER chimeric gene [Cell, 54, 199 (1988);Proc. Natl. Acad. Sci., USA, 90, 1657 (1993)] (apportioned from Dr.Shigeaki Kato, University of Tokyo) was cleaved with AatII and NdeI andsubjected to a Klenow treatment to prepare an AatII (blunt end)-NdeI(blunt end) fragment.

The above mentioned PvuII (blunt end)-EcoRI (blunt end) fragment derivedfrom pSV2bsr and the AatII (blunt end)-NdeI (blunt end) fragment derivedfrom ERαAF2 in pM were ligated to construct a plasmid pGERbsrR2.pGERbsrR2 is able to express a chimeric protein (Gal4-ER) consisting ofa DNA binding domain of transcription factor Gal4p derived from a yeast(Saccharomyces cerevisiae) and a ligand binding domain of estrogenreceptor.

(2) Construction of an Inducible Expression Plasmid of FireflyLuciferase

pcDNA3 (Invitrogen) was cleaved with XhoI and subjected to a Klenowtreatment to prepare a XhoI (blunt end) fragment. The fragment wasligated to construct pcDNA3 where cleaved sites by XhoI disappeared.pcDNA3 where the cleaved site with XhoI disappeared was cleaved withKpnI and subjected to a Klenow treatment to prepare a KpnI (blunt end)fragment. The fragment was ligated to construct pcDNA3 where cleavedsites with XhoI and KpnI disappeared. The plasmid was cleaved with BglIIand subjected to a Klenow treatment to a prepare BglII (blunt end)fragment.

pAMoERC3Sc (Japanese Published. Unexamined Patent Application No.336,963/1993) was cleaved with XhoI and NsiI and subjected to a Klenowtreatment to obtain a XhoI (blunt end)-NsiI (blunt end) fragment of 2.2kb having an oriP sequence.

The above-mentioned BglII (blunt end) fragment derived from pcDNA3 whereXhoI-cleaved site and KpnI-cleaved site disappeared and the XhoI (bluntend)-NsiI (blunt end) fragment derived from pAMoERC3Sc were ligated toconstruct a plasmid pcDNA3-oriP. pcDNA3-oriP was cleaved with XhoI andHindIII to obtain a XhoI-HindIII fragment.

pSE0luc2 (WO 98/14474) was cleaved with XhoI and NcoI and subjected to aKlenow treatment to obtain a XhoI (blunt end)-NcoI (blunt end) fragmentcomprising the ampicillin-resistant gene. The fragments were ligated toconstruct a plasmid pASd1-luc1. After pASd1-luc1 was cleaved with XhoIand HindIII, a XhoI-HindIII fragment of 0.11 kb were obtained.

The above-mentioned XhoI-HindIII fragment derived from pcDNA3-oriP andthe XhoI-HindIII fragment derived from pASd1-luc1 were ligated toconstruct a plasmid pcDNA3-oriP-Sd1. pcDNA3-oriP-Sd1 was cleaved withXhoI and KpnI to obtain a XhoI-KpnI fragment.

Four kinds of DNAs having nucleotide sequences represented by SEQ IDNOS: 1, 2; 3 and 4, respectively were synthesized by DNA synthetizer.When the synthetic DNAs were mixed and annealed, a double-stranded DNAhaving polyadenylation signal was constructed. Each of the syntheticDNAs was phosphorylated using T4 polynucleotide kinase, mixed andannealed to give a double-stranded DNA.

When the double-stranded DNA was ligated to the XhoI-KpnI fragmentderived from pcDNA3-oriP-Sd1, a plasmid pcDNA3-oriP-Sd1-pA wasconstructed. pcDNA3-oriP-Sd1-pA was cleaved with XhoI and subjected to aKlenow treatment to obtain a XhoI (blunt end) fragment.

pFR-luc (manufactured by Stratagene) was cleaved with HindIII and BamHIand subjected to a Klenow treatment to obtain a HindIII (bluntend)-BamHI (blunt end) fragment of 0.14 kb.

The above-mentioned XhoI (blunt end) fragment derived frompcDNA3-oriP-Sd1-pA and the HindIII-BamHI fragment derived from pFR-lucwere ligated to obtain a plasmid pAGalSd1. The pAGalSd1 comprises apromoter having a sequence where Gal4p-responsive elements (UASG) arerepeated for 5 times. pAGalSd1 was cleaved with EcoRI and subjected to aKlenow treatment to obtain a EcoRI (blunt end) fragment.

pSE0luc2 (WO 98/14474) was cleaved with HindIII and SacI and subjectedto a Klenow treatment to prepare a HindIII (blunt end)-SacI (blunt end)fragment of 1.7 kb comprising the firefly luciferase gene.

The above-mentioned HindIII (blunt end)-SacI (blunt end) fragmentsderived from pSE0luc2 and the EcoRI (blunt end) derived from pAGalSd1were ligated to construct a plasmid pAGalSd1-luc.

Among the two HindIII sites existing in pAGalSd1-luc, only a HindIIIsite far from the firefly luciferase gene was made disappeared by aKlenow treatment to construct pAGalSd4-luc.

pAGalSd4-luc was cleaved with Asp718 and subjected to a partialdigestion with StuI to obtain an Asp718-StuI fragment of 9.5 kb, derivedfrom pAGalSd4-luc. The DNA fragments were subjected to a Klenowtreatment and self-ligated to construct a plasmid pAGal9-luc.

(3) Construction of Inducible Expression Vectors pAGal9-d and pAGal9-nd

Expression plasmid pAGal9-luc having oriP of Epstein-Barr virus wascleaved with HindIII and SacI to prepare a HindIII-SacI fragment of 6.9kb containing oriP.

pAMo-d (Japanese Published Unexamined Patent Application No.211,885/2001) was cleaved with HindIII and SacI to prepare aHindIII-SacI fragment comprising the tetracycline-resistant gene(Tc^(R)).

The above-mentioned HindIII-SacI fragment derived from pAGal9-luc andthe HindIII-SacI fragment derived from pAMo-d were ligated to constructa plasmid pAGal9-d where the firefly luciferase gene in pAGal9-luc wassubstituted with a stuffer sequence of pAMo-d. pAGal9-luc was cleavedwith HindIII and SacI to prepare a HindIII-SacI fragment of 6.9 kb.

pAMo-nd (Japanese Published Unexamined Patent Application No.211,885/2001) was cleaved with HindIII and SacI to prepare aHindIII-SacI fragment comprising the tetracycline-resistant gene.

The above-mentioned HindIII-SacI fragment derived from pAGal9-luc andthe HindIII-SacI fragment derived from pAMo-nd were ligated to constructa plasmid pAGal9-nd where the firefly luciferase gene in pAGal9-luc wassubstituted with a stuffer sequence of pAMo-nd.

(4) Preparation of a Cell Line KJMGER8 Where Gal4-ER Expression PlasmidpGERbsrR2 was Integrated in Chromosomal DNA of Namalwa KJM-1 Cells

Gal4-ER chimeric transcription factor expression plasmid pGERbsrR2 wasdissolved in a TE buffer [10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L ofethylenediamine tetraacetate] so as to make 1 μg/μL and, after that, theplasmid, 4 μg for 6×10⁶-cells, was transfected to Namalwa KJM-1 cells[Cytotechnology, 1, 151 (1988)] by an electroporation method[Cytotechnology, 3, 133 (1990)] to prepare transformed cells. NamalwaKJM-1 cell is a B-cell line adapted for serum-free culture, and capableof expressing the EBNA-1 gene.

The transformant was suspended in 8 ml of an RPMI 1640-ITPSG medium [amedium where a 1/40 amount of 7.5% NaHCO₃, 3% 200 mmol/L of L-glutaminesolution (manufactured by Invitrogen), 0.5% penicillin-streptomycinsolution (manufactured by Invitrogen comprising 5,000 units/ml ofpenicillin and 5,000 μg/ml of streptomycin), 10 mmol/L ofN-2-hydroxyethylpiperazine-N′-2-hydroxypropane-3-sulfonic acid (HEPES),3 μg/ml insulin, 5 μg/ml transferrin, 5 mmol/L sodium pyruvate, 125nmol/L sodium selenite and 1 mg/ml galactose were added to an RPMI 1640medium (manufactured by Nissui Seiyaku)] and cultured at 37° C. in a CO₂incubator for 24 hours.

After the cultivation, blasticidin S (KK-400: manufactured by KakenSeiyaku) was added so as to make 2.0 μg/ml, dispensed in a 96-well plate(500 to 2,000 cells/well) and cultivation was carried out to obtain manystable transformants (single clones) where pGERbsrR2 was integrated inchromosomal DNA. Each transformant was subcultured in RPMI 1640-ITPSGmedium containing 2.0 μg/ml of blasticidin S.

By the method as mentioned below, an excellent stable transformantKJMGER8 cell having high induction ratio and low background uponnon-inducing stage was selected from the above-mentioned stabletransformants.

An inducible expression plasmid pAGalSd1-luc of firefly luciferase wastransfected to each transformant by an electroporation method andcultured for 2 days.

After the cultivation, 17β-estradiol (E8875: manufactured bySigma)(final concentration 10 nmol/L) was added and, after thecultivation for 24 hours more, the firefly luciferase activity wasmeasured. For the measurement of the activity, a luminometer-LB 953(manufactured by Berthold) was used, 100 μl of a buffer for dissolvingthe cells [1% Triton X-100, 100 mmol/L KH₂PO₄ (pH 7.8) and 1 mmol/Ldithiothreitol] was automatically injected into the above culturesolution, then 300 μl of a substrate solution [25 mmol/L glycylglycine(pH7.8), 15 mmol/L MgSO₄, 5 mmol/LATP and 0.33 mmol/L luciferin] wasautomatically injected and the amounts of emission of light during 10seconds was measured for adopting as a luciferase activity. Forcomparison, luciferase activity under the condition where no17β-estradiol was added was also measured.

Luciferase activity under the condition where 17β-estradiol was addedand luciferase activity under the condition where no 17β-estradiol wasadded were compared, induction ratio for gene expression was calculated,and KJMGER8 cell was selected as a clone with high induction ratio andlow luciferase activity in the condition without addition of17β-estradiol.

REFERENTIAL EXAMPLE 59 Construction of a Reporter Plasmid pACREplucWhere Firefly Luciferase is a Reporter

pACREpluc which is a reporter plasmid capable of expressing a fireflyluciferase gene under the control of cAMP-responding element (CRE) wasconstructed by the following method. pACREpluc has oriP of Epstein Barrvirus and the hygromycin-resistant gene.

pAMo [J. Biol. Chem., 268, 22782 (1993); another name: pAMoPRC3Sc(Japanese Published Unexamined Patent Application No. 336,963/1993)] waspartially digested with ClaI to obtain a DNA fragment where one site wascleaved. The DNA fragment was partially digested by MluI to obtain aClaI-MluI fragment of 9.5 kb. pAGE248 [J. Biol. Chem., 269, 14730(1994)] was cleaved with ClaI and MluI to obtain a ClaI-MluI fragment of1.5 kb comprising the hygromycin-resistant gene. The ClaI-MluI fragmentderived from pAMo and the ClaI-MluI fragment derived from pAGE248 wereligated to construct a plasmid pAMoh.

pAMoh was cleaved with XhoI and HindIII to obtain a XhoI-HindIIIfragment comprising the hygromycin-resistant gene. pAGal9-luc wascleaved with SalI and HindIII to obtain a SalI-HindIII fragmentcomprising oriP and Gal4UAS. The SalI-HindIII fragment derived frompAGal9-luc and the above-mentioned XhoI-HindIII fragment derived frompAMoh were ligated to construct a plasmid pAGal9h.

pBluescriptII KS+ (manufactured by Toyoboseki) was cleaved with SalI andXhoI and subjected to a dephosphorylation using phosphatase (AlkalinePhosphatase E. coli C75; manufactured by Takara Shuzo) to obtain aSalI-XhoI fragment comprising the ampicillin-resistant gene. As a resultof annealing of synthetic oligonucleotides having nucleotide sequencesof SEQ ID NOs: 5 and 6, respectively, a double-stranded DNA containingtwo CRE sequences was prepared. The double-stranded DNA was ligated tothe above SalI-XhoI fragment derived from pBluescriptII KS+ to constructa plasmid pBS-CREI comprising two CRE sequences. The pBS-CREI is aplasmid where the double-stranded DNA is inserted in such a directionthat cleaved site with SalI and cleaved site with XhoI are regeneratedand has each one of the above cleaved sites.

pBS-CREI was cleaved with ScaI and XhoI to prepare a ScaI-XhoI fragmentcomprising ori of a phage f1. pBS-CREI was cleaved with ScaI and SalI toprepare a ScaI-SalI fragment comprising Co1E1 ori. The ScaI-XhoIfragment and the ScaI-SalI fragment derived from pBS-CREI were ligatedto construct pBS-CREII comprising 4 CRE sequences.

pBS-CREII was cleaved with ScaI and XhoI to obtain a ScaI-XhoI fragmentcomprising ori of a phage f1. pBS-CREII was cleaved with ScaI and SalIto obtain a ScaI-SalI fragment comprising ColE1 ori. The ScaI-XhoIfragment and the ScaI-SalI fragment derived from pBS-CREII were ligatedto construct pBS-CREIV comprising 8 CRE sequences.

pBS-CREIV was cleaved with ScaI and XhoI to obtain a ScaI-XhoI fragmentcomprising ori of a phage f1. pBS-CREIV was cleaved with ScaI and SalIto obtain a ScaI-SalI fragment comprising Co1E1 ori. The ScaI-XhoIfragment and the ScaI-SalI fragment derived from pBS-CREIV were ligatedto construct PBS-CREVIII comprising 16 CRE sequences.

pBS-CREVIII was cleaved with XhoI, subjected to a Klenow treatment, andfurther cleaved with HindIII to obtain a HindIII-XhoI (blunt end)fragment comprising 16 CREs. pAGalSd1 was cleaved with MluI and HindIIIto obtain a MluI-HindIII fragment of 1.4 kb. pAGal19h was cleaved withXbaI, subjected to a Klenow treatment, and further cleaved with MluI togive a XbaI (blunt end)-MluI fragment. The HindIII-XhoI (blunt end)fragment derived from pBS-CREVIII, the MluI-HindIII fragment derivedfrom pAGalSd1 and the XbaI (blunt end)-MluI fragment derived frompAGal19h were ligated to prepare a plasmid pACREh.

pAGal9-luc was cleaved with XhoI and NotI to obtain a XhoI-NotI fragmentcomprising the firefly luciferase gene. pACREh was cleaved with XhoI andNotI to obtain a XhoI-NotI fragment comprising CRE sequences. TheXhoI-NotI fragment derived from pAGal9-luc and the XhoI-NotI fragmentderived from pACREh were ligated to construct a plasmid pACREluc.

pACREluc was cleaved with HindIII, subjected to a Klenow treatment, andfurther cleaved with XhoI to obtain a HindIII (blunt end)-XhoI fragmentcomprising CRE and a HindIII (blunt end)-XhoI fragment comprising thefirefly luciferase gene, respectively. The above-mentioned two HindIII(blunt end)-XhoI fragments derived from pACREluc were ligated toconstruct a plasmid pACRElucH in which HindIII site in upstream of CREsequence in pACREluc disappeared.

pGL3-Enhancer vector (manufactured by Promega) was cleaved with HindIIIand HpaI to obtain a HindIII-HpaI fragment comprising the luc+ gene (animproved firefly luciferase gene). pACRElucH was cleaved with NotI,subjected to a Klenow treatment, and further cleaved with HindIII toobtain a HindIII-NotI (blunt end) fragment containing CRE. TheHindIII-HpaI fragment derived from pGL3-Enhancer vector and theHindIII-NotI (blunt end) fragment derived from pACRElucH were ligated toconstruct a plasmid pACREpluc.

REFERENTIAL EXAMPLE 60 Construction of a Plasmid with InducibleExpression of GPR4

1 μg of mRNA derived from human lung (manufactured by Clontech) was usedand a single-stranded cDNA was synthesized using a SUPERSCRIPTFirst-Stranded Synthesis System for RT-PCR (manufactured by Gibco). GPR4cDNA was prepared by PCR using a solution (5 μl) where saidsingle-stranded cDNA was diluted with water to an extent of 250-fold asa template and synthetic DNA having nucleotide sequences represented bySEQ ID NO: 7 and NO: 8 as a GPR4 gene-specific primer. Sequence of theGPR4 gene-specific primer was designed on the basis of sequenceinformation of GPR4 gene (GenBank Accession No. U21051). With regard toan enzyme, PfuTurbo DNA Polymerase (manufactured by Stratagene) wasused. With regard to a buffer for conducting the PCR, a buffer of10-fold concentration attached to the enzyme was used. The PCR wasconducted by treating at 95° C. for 5 minutes and conducting 30 cyclesof reaction each comprising at 94° C. for 1 minute, at 60° C. for 1minute and at 72° C. for 1 minute using a thermal cycler DNA engine(manufactured by MJ Research).

The amplified GPR4 cDNA fragment was cleaved with HindIII and NotI whichcleave a sequence designed on a primer. Fragment containing GPR4 cDNAwas recovered by an agarose gel electrophoretic method.

Said cleaved fragment was integrated between HindIII and NotI of plasmidpAGal9-nd whereupon a plasmid pAGal9-GPR4 with inducible expression ofGPR4.

Nucleotide of sequences of 5′-end and 3′-end of said cDNA weredetermined using a primer (synthetic. DNA having sequences representedby SEQ ID NO: 9 and NO: 10) which is specific to nucleotide sequence inpAGal9-nd. A synthetic DNA which is specific to the determinednucleotide sequence was prepared and, when it was used as a primer, anucleotide sequence farther ahead was determined. As a result ofrepeating said method, total nucleotide sequence of said cDNA wasdetermined and it was confirmed to code for GPR4. For the determinationof the nucleotide sequence, a DNA sequencer 377 of Perkin Elmer and areaction kit (ABI Prism™ BigDye™ Terminator Cycle Sequencing ReadyReaction kit: Applied Biosystems) were used.

A nucleotide sequence of DNA fragment integrated into a plasmid wasdetermined and it was confirmed to code for GPR4.

REFERENTIAL EXAMPLE 61 Construction of Assay Cells of GPR4

2 μg of plasmid pAGal9-GPR4 with inducible expression of GPR4 and 2 μgof reporter plasmid pACREpluc were co-transferred into 6×10⁶ cell ofKJMGER8 by the above-mentioned electroporation. The transformants weresuspended in 8 ml of an RPMI 1640-ITPSG medium and cultured in a CO₂incubator at 37° C. for 24 hours. After the cultivation, blasticidin S(2.0 μg/ml), hygromycin B (300 μg/ml) and geneticin (500 μg/ml) wereadded and cultivation was carried-out for 14 days more, and stabletransformants (called GPR4 assay cells) were obtained. The transformantswere subcultured in an RPMI 1640-ITPSG medium containing blasticidin S(2.0 μg/ml), hygromycin B (300 μg/ml) and geneticin (500 μg/ml).

Similarly, control plasmid pAGal9-nd (2 kg) and reporter plasmidpAGREpluc (2 μg) were co-transferred into KJMGER8 and a stabletransformant (called control cell) was obtained.

REFERENTIAL EXAMPLE 62 Cloning of DNA Coding for Human GPR4 HomologDerived from Mice

Based upon the nucleotide sequence information of human GPR4 gene[Accession (AC) No. U21051], search was conducted using a database ofNCBI as an object. As a result, mouse genome sequence (AC 073784) andplural expression sequence tag (EST) sequences (BF 178464, AA 968193, AA798732, AI 840893 and AI 851037) were selected as sequences having highhomology. A nucleotide-sequence of a gene constructed from said mousegenome sequence and EST is shown in SEQ ID NO: 14 and an amino acidsequence of a polypeptide encoded by said gene is shown in SEQ ID NO:13. When said amino acid was compared with the amino acid sequence ofhuman GPR4 using an analysis program [GENETYX WIN ver. 5.0 (manufacturedby Software)], it is confirmed that an identity score is 92.7%.

Therefore, it is presumed that a polypeptide having the amino acidsequence represented by SEQ ID NO: 13 is a human GPR4 homolog of mouse(mouse GPR4).

Accordingly, DNA coding for mouse GPR4 can be prepared by a PCR wheremouse cDNA library which is commercially available or can be prepared bya known method is used as a template and an oligonucleotide which isable to be designed and synthesized depending upon the nucleotidesequence represented by SEQ ID NO: 14 as a primer set.

REFERENTIAL EXAMPLE 63 Cloning of DNA Coding for a Human GPR4 HomologDerived from Rats

Based upon the nucleotide sequence information of human GPR4 gene (ACNo. U21051), search was conducted using a database of NCBI as an object.As a result, two rat genome sequences (AC 119447.2 and AC 096180.2) andplural rat EST sequences (BF 544182, AI 170948, AI-008858, AI 235374, AI502871 and BQ 194515) were selected as sequences having high homology.Based upon those sequences and the nucleotide sequence information ofmice shown by SEQ ID NO: 14, oligonucleotides having the nucleotidesequences represented by SEQ ID NO: 15 and SEQ ID NO: 16 were prepared.

Each 1.0 μmol/L of said oligonucleotides was used as a primer set and 2μL of cDNA prepared from mRNA derived from rat lung was used as atemplate whereby, in order to make the concentration of each componentwhich is mentioned below 200 μmol/L, 40 μL of a reaction solutioncontaining 2.5 units of dNTP (dATP, dGTP, dCTP, dTTP), TagGold(manufactured by Perkin Elmer) and 1×Taq Gold (Mg plus) buffer (PerkinElmer) was prepared and then a PCR was conducted under the followingcondition.

Thus, a thermal cycler PTC-200 (manufactured by MJ Research) was usedand heating was conducted at 95° C. for 10 minutes, then 30 cycles ofreaction each comprises 94° C. for 1 minute, 55° C. for 1 minute and 72°C. for 1 minute were conducted and, further, heating at 72° C. for 5minutes was conducted.

From the resulting reaction solution of the PCR, 5 μL was collected and,after confirming by agarose gel electrophoresis that about 1.1 kb of DNAfragments presumed to be DNA coding for GPR4 were amplified, DNAfragments were eluted and recovered using QIAEX II Gel Extraction Kit(manufactured by Qiagen) according to the manual attached to the kit.

The above-recovered DNA fragments (50 ng) and 50 ng of pT7Blue T-Vector(manufactured by Novagen) were ligated using DNA Ligation Kit ver. 2(manufactured by Takara Shuzo) according to the manual attached to thekit to prepare a recombinant plasmid DNA. A plasmid pT7RG was preparedby a conventional method from a transformant obtained by atransformation of Escherichia coli JM109 strain using the resultingrecombinant plasmid DNA. As a result of determination of the totalnucleotide sequence of the plasmid pT7RG, the pT7RG contains about 1.1kb of cDNA having the nucleotide sequence represented by SEQ ID NO: 18.An amino acid sequence of a polypeptide corresponding to DNA comprisingthe nucleotide sequence represented by SEQ ID NO: 18 is shown in SEQ IDNO: 17. When the amino acid sequence was compared with amino acidsequences of human and mouse GPR4 using an analysis program [GENETYX WINver. 5.0 (manufactured by Software)], it is confirmed that identityscores are 93.0% and 99.2%, respectively.

Consequently, it has been clarified that a polypeptide having the aminoacids represented by SEQ ID NO: 17 is a human GPR4 homolog of rat (ratGRP4).

EXAMPLE 1 Tablet

A tablet comprising the following composition is prepared by aconventional method. Formulation Compound 1 20 mg Lactose 143.4 mg  Potato starch 30 mg Hydroxypropyl cellulose  6 mg Magnesium stearate 0.6mg  200 mg 

EXAMPLE 2 Injection Solution

Injection solution comprising the following composition is prepared by aconventional method. Formulation Compound 5 2 mg Pure soybean oil 200 mgPure egg yolk lecithin 24 mg Glycerol for injection 50 mg Distilledwater for injection 1.72 ml 2.00 ml

INDUSTRIAL APPLICABILITY

The present invention provides an agent for prevention and/or treatmentof asthma comprising a substance capable of suppressing the functioninvolved in signal transduction of GPR4 as an active ingredient; anagent for prevention and/or treatment of asthma which comprises anitrogen-containing tricyclic compound or a quaternary ammonium saltthereof, or a pharmaceutically acceptable salt thereof as an activeingredient.

Free Text of Sequence Listing

SEQ ID NO: 1—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 2—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 3—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 4—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 5—Illustration of artificial sequence: Synthetic DNA

SEQ ID. NO: 6—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 7—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 8—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 9—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 10—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 15—Illustration of artificial sequence: Synthetic DNA

SEQ ID NO: 16—Illustration of artificial sequence: Synthetic DNA

1. An agent for prevention and/or treatment of asthma, which comprises,as an active ingredient, a substance capable of suppressing the functioninvolved in signal transduction of a protein having the amino acidsequence represented by SEQ ID NO:
 11. 2. An agent for prevention and/ortreatment of asthma, which comprises one of the following 1) to 4) as anactive ingredient: 1) an oligonucleotide having a sequence complementaryto that of oligonucleotide comprising continuous 5 to 60 nucleotidesselected from the nucleotide sequence represented by SEQ ID NO: 12 or aderivative of said oligonucleotide, 2) an oligonucleotide having asequence complementary to that of oligonucleotide comprising continuous5 to 60 nucleotides selected from the nucleotide sequence represented bySEQ ID NO: 14 or a derivative of said oligonucleotide, 3) anoligonucleotide having a sequence complementary to that ofoligonucleotide comprising continuous 5 to 60 nucleotides selected fromthe nucleotide sequence represented by SEQ ID NO: 18 or a derivative ofsaid oligonucleotide, and 4) an oligonucleotide comprising 5 to 60nucleotides which hybridizes under stringent conditions with DNA havingthe nucleotide sequence represented by one member selected from SEQ IDNOs: 12, 14 and 18 and which is capable of suppressing the functioninvolved in signal transduction of protein having the amino acidsequence represented by SEQ ID NO: 11 or a derivative of saidoligonucleotide.
 3. An agent for prevention and/or treatment of asthma,which comprises one of the following 1) to 4) as an activeingredient: 1) an antibody which recognizes a protein having the aminoacid sequence represented by SEQ ID NO: 11, 2) an antibody whichrecognizes a protein having the amino acid sequence represented by SEQID NO: 13, 3) an antibody which recognizes a protein having the aminoacid sequence represented by SEQ ID NO: 17, and 4) an antibody, whichrecognizes a protein having the amino acid sequence in which one or moreamino acid(s) is/are deleted, substituted or added in the amino acidsequence represented by one member selected from SEQ ID NOs:11, 13 and17 and which has the function involved in signal tranduction of aprotein having the amino acid sequence represented by SEQ ID NO:11. 4.An agent for prevention and/or treatment of asthma, which comprises anitrogen-containing tricyclic compound represented by the formula (I) ora quaternary ammonium salt thereof, or a pharmaceutically acceptablesalt thereof;

[wherein R¹ represents a substituted or unsubstituted heterocyclicgroup, —NR⁵R⁶ (wherein R⁵ and R⁶ are the same or different and eachrepresents hydrogen, substituted or unsubstituted lower alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedlower alkenyl, substituted or unsubstituted lower alkynyl, substitutedor unsubstituted aralkyl or substituted or unsubstituted heterocyclicalkyl, or R⁵ and R⁶ are combined together with the adjacent nitrogenatom to form a substituted or unsubstituted heterocyclic group), —OR⁷(wherein R⁷ represents hydrogen, substituted or unsubstituted loweralkyl, substituted or unsubstituted lower alkanoyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted lower alkenyl,substituted or unsubstituted lower alkynyl, substituted or unsubstitutedaryl, substituted or unsubstituted aralkyl or substituted orunsubstituted heterocyclic alkyl), —SR^(7a) (wherein R^(7a) has the samemeaning as the above R⁷), —CONR^(5a)R^(6a) (wherein R^(5a) and R^(6a)have the same meanings as the above R⁵ and R⁶, respectively), —CO₂R^(7b)(wherein R^(7b) has the same meaning as the above R⁷), —N⁺R^(5b)R^(6b)R⁸(wherein R^(5b) and R^(6b) have the same meanings as the above R⁵ andR⁶, respectively, and R⁸ represents lower alkyl, lower alkenyl oraralkyl), formyl, carboxy or cyano; R² represents hydrogen, substitutedor unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted lower alkenyl, substituted or unsubstitutedlower alkynyl, substituted or unsubstituted aralkyl or substituted orunsubstituted heterocyclic alkyl; R³ and R⁴ are the same or differentand each represents hydrogen, lower alkyl or halogen; n represents 0 or1; X represents —(CH₂)₂— or —CH═CH—; and Y represents the formula (II);

(wherein W represents CH or a nitrogen atom; Z¹ and Z² are the same ordifferent and each represents hydrogen, substituted or unsubstitutedlower alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted lower alkenyl, substituted or unsubstituted lower alkynyl,substituted or unsubstituted aryl, substituted or unsubstituted aralkylor substituted or unsubstituted heterocyclic alkyl, or Z¹ and Z² arecombined together with two carbon atoms being adjacent to each of themto form a substituted or unsubstituted aromatic ring or substituted orunsubstituted heterocycle; and Z³ represents hydrogen, substituted orunsubstituted lower alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted lower alkenyl, substituted or unsubstitutedlower alkynyl, substituted or unsubstituted aryl, a substituted orunsubstituted heterocyclic group, substituted or unsubstituted aralkylor substituted or unsubstituted heterocyclic alkyl)] as an activeingredient.
 5. The agent for prevention and/or treatment of asthmaaccording to claim 4, wherein R¹ is —NR⁵R⁶ and R⁵ and R⁶ are combinedtogether with the adjacent nitrogen atom to form a substituted orunsubstituted heterocyclic group.
 6. The agent for prevention and/ortreatment of asthma according to claim 4, wherein R² is hydrogen.
 7. Theagent for prevention and/or treatment of asthma according to claim 4,wherein R3 and R4 are hydrogen.
 8. The agent for prevention and/ortreatment of asthma according to claim 4, wherein Z1 and Z2 are combinedtogether with two carbon atoms being adjacent to each of them to formsubstituted or unsubstituted heterocycle.
 9. A method for preventionand/or treatment of asthma, which comprises administering an effectiveamount of the nitrogen-containing tricyclic compound or the quaternaryammonium salt thereof, or the pharmaceutically acceptable salt thereofdescribed in claim
 4. 10. Use of the nitrogen-containing tricycliccompound or the quaternary ammonium salt thereof, or thepharmaceutically acceptable salt thereof described in claim 4 for themanufacture of an agent for prevention and/or treatment of asthma.
 11. Amethod for prevention and/or treatment of asthma, which comprisesadministering a therapeutically effective amount of a substance capableof suppressing the function involved in signal transduction of a proteincomprising the amino acid sequence represented by SEQ ID NO:
 11. 12. Amethod for prevention and/or treatment of asthma, which comprisesadministering a therapeutically effective amount of an oligonucleotideor a derivative of said oligonucleotide which is any one of 1) to 4)described in claim
 2. 13. A method for prevention and/or treatment ofasthma, which comprises administering a therapeutically effective amountof an antibody which is any one of 1) to 4) described in claim
 3. 14.Use of a substance capable of suppressing the function involved insignal transduction of a protein having the amino acid sequencerepresented by SEQ ID NO: 11 for the manufacture of an agent forprevention and/or treatment of asthma.
 15. Use of an oligonucleotide ora derivative of said oligonucleotide which is any one of 1) to 4)described in claim 2 for the manufacture of an agent for preventionand/or treatment of asthma.
 16. Use of an antibody which is any oneof 1) to 4) described in claim 3 for the manufacture of an agent forprevention and/or treatment of asthma.